S.agalactiae antigens I + II

ABSTRACT

The present invention discloses isolated nucleic acid molecules encoding a hyperimmune serum reactive antigen or a fragment thereof as well as hyperimmune serum reactive antigens or fragments thereof from  S. agalactiae , methods for isolating such antigens and specific uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. §371 ofInternational Application No. PCT/EP2004/004856 filed 6 May 2004, whichclaims priority to European Patent Application No. 03450112.2 filed 7May 2003 and European Patent Application No. 03450266.6 filed 28 Nov.2003. The entire text of each of the above-referenced disclosures isspecifically incorporated by reference herein without disclaimer.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING SUBMITTED ON A COMPACTDISC

The Sequence Listing is submitted on one compact disc (Copy 1), togetherwith a duplicate thereof (Copy 2), each created on Nov. 7, 2005, andeach containing one 1,161 kb file entitled “SONN080.APP.txt.” Thematerial contained on the compact disc is specifically incorporatedherein by reference.

The present invention relates to isolated nucleic acid molecules, whichencode antigens for Streptococcus agalactiae, which are suitable for usein preparation of pharmaceutical medicaments for the prevention andtreatment of bacterial infections caused by Streptococcus agalactiae.

Streptococcus agalactiae is a gram-positive bacterium, and belongs tothe Group B Streptococci (GBS) based on its hemolysis pattern in bloodagar. The organism is encapsulated, and capsule is an important elementof pathogenicity. Capsules are antigenic and form the basis forclassifying GBS by serotypes. Nine distinct GBS serotypes have beenidentified: Ia, Ib, II, III, IV, V, VI, VII and VIII. Most S. agalactiaeserotypes have been shown to cause serious disease, and the two mostcommon serotypes—type III and V—are estimated to account for themajority of invasive disease worldwide. The ranking and serotypeprevalence differs by age group and geographic area. In the US, GBS typeIII causes more than 50% of infant disease, type V about 40% ofnonpregnant adult disease, and type Ia about a third of disease in anypatient population.

Streptococcus agalactiae is an important agent of human disease at theextremities of age and in those who have underlying disease. Group BStreptococci are the major cause of generalized and focal infections inthe newborn infant. GBS is the predominant pathogen in newborns in theUS since the 1970's. Bacterial infection can lead to life threateningdiseases, such as sepsis, pneumonia and meningitis. Survivors can becomepermanently handicapped with hearing, learning and visual disabilities.Newborns usually acquire the organism intrapartum or during deliveryfrom their GBS-colonized mothers. In addition, GBS are also a frequentcause of infections in pregnant women and in chronically ill and elderlypatients, such as those suffering from diabetes, malignancies,immunodeficiencies, etc., (reviewed by Balter, S. et al. in Grampositive pathogens ed. by Fischetti V. A. et al. ASM Press 2000, pp154-160).

10-35% of pregnant women are colonized with GBS, but are asymptomatic.However, GBS colonization is important because of the risk of verticaltransmission. 50-70% of neonates born to colonized women—that is 5-15%of all newborns—become colonized by GBS during delivery. Colonization isa prerequisite for infection and disease. The most preterm infants areat the highest risk for invasive disease due to low maternal transfer ofantibodies and immature immune system. GBS carriage during pregnancy maybe chronic, intermittent, or transient. It is difficult to assess theduration of carriage, since women are screened once during a prenatalvisit from the late trimester. Several studies suggest that there is acorrelation (˜90%) between the colonization status in the thirdtrimester and at the time of delivery. Serotyping of the isolatesindicates that persistence of the same type is most common (reviewed byBalter, S. et al. in Gram positive pathogens ed. by Fischetti V. A. etal. ASM Press 2000, pp 154-160).

Without preventive intervention, 1 to 2% of all neonates and 15% ofneonates born to heavily colonized women develop invasive disease(sepsis, pneumonia and/or meningitis). In the US, GBS infections affect1-5 newborns/1000 live birth. About 17.000 cases of invasive GBS diseaseoccurred in the US annually, and 7.500 occurred in newborns beforeprevention. GBS is the most common cause of bacterial meningitisaccounting for ˜40% of all cases reported in this age group. The overallincidence of invasive GBS disease is 0.2-0.7/100.000 in the US. It issomewhat lower in Europe. Mortality without preventive intervention is6% with invasive disease, every 16. infected newborn dies and 20% ofsurvivors become permanently handicapped.

The rates of serious group B strep infections are much higher amongnewborns than among any other age group. Nonetheless, serious group Bstrep infections occur in other age groups in both men and women. Amongnon-pregnant adults, rates of serious disease range from 4.1 to 7.2cases per 100,000 populations. The average death rate for invasiveinfections (infections where the bacteria have entered a part of thebody that is normally not exposed to bacteria) is 8-10% for adult's ages18-64 and 15-25% for adults 65 years of age and over. Mortality ratesare lower among younger adults, and adults who do not have other medicalconditions. The rate of serious group B strep disease increases withage. The average age of cases in non-pregnant adults is about 60 yearsold. Most adult group B strep disease occurs in adults who have seriousmedical conditions. These include: diabetes mellitus; liver disease;history of stroke; history of cancer; or bedsores. Among the elderly,rates of serious group B strep disease are more common among residentsof nursing facilities, and among bedridden hospitalized patients. GroupB strep disease among non-pregnant adults may often be acquired afterrecent trauma, or after having certain invasive hospital procedures likesurgery ({Farley, M., 2001}; {Jackson, L. et al., 1995};www.cdc.gov/groupbstrep/).

Direct medical costs of neonatal disease before prevention were $294million annually and GBS continues to pose a considerable economicburden.

A definitive diagnosis of infection with Streptococcus agalactiaegenerally relies on isolation of the organism from cervical swabs, bloodor other normally sterile body sites. Tests are also available to detectcapsular polysaccharide antigen in body fluids.

Penicillin G is the treatment of choice for established cases of GBS.Ten days of treatment is recommended for bacteremia, pneumonia and softtissue infections, while 2-3 weeks is recommended for meningitis and 3-4weeks for osteomyelitis.

Prevention has been established since 1994 in North America by screeningpregnant women for carriage of GBS, taking vaginal and anorectal swabsat 35-37 weeks' gestation, or by identifying risk factors at admissionfor delivery without cultures. Women who are candidates for prophylaxisare given intrapartum antibiotic therapy during labor to preventearly-onset neonatal disease. This prevention method has decreased theincidence of GBS disease from 1.7 to 0.4/1000 live births between 1993and 1999 in the US. Although most neonatal GBS disease can be preventedthrough intrapartum prophylaxis (Penicillin G or Ampicillin), currentlyavailable strategies are not ideal, especially for the prevention oflate-onset (>7 days of age) infections and disease in premature babies.There are always individuals who escape of screening for carriage due toseveral reasons, such as intermittent carriers, who are tested negativeat wks 32-35, but become positive during delivery, unattendance,negligence, or delivery before screening date (32-35 wks).

In the long run, widespread use of antibiotics usually induces resistantstrains that appear after a period of time. Extensive use of Penicillin(every 3-5^(th) women are treated with high dose), and other antibioticshas already been shown to steadily increase the percentage of antibioticresistant clinical isolates (ref). Moreover, efficiency of antibioticbased prevention is not that effective for late onset disease, as it isfor early onset (within 48 hrs after delivery). An additional concern isthat prevention in susceptible adult populations has not been addressed.

Vaccine development is hindered by the lack of sufficient knowledgeabout the elements of protective immunity against GBS carriage anddisease. The relationship of carriage to the development of naturalimmunity is poorly understood. In addition, the immunologic mechanismthat allows disease to occur in a carrier is ill defined. However, it issuggested that the maternal serum levels of pathogen-specific antibodiesare correlated with neonatal GBS disease. It has been firmly establishedthat there is an inverse correlation between maternal anti-capsularpolysaccharide antibody levels at delivery and the frequency of invasiveneonatal diseases {Campbell, J. et al., 2000}.

Although the group B carbohydrate antigen is common to all strains ofGBS, unfortunately, it is not strongly immunogenic and antibodies arenot protective from lethal challenge in experimental models. The GBScapsule itself that is made of polysaccharides, is immunogenic and isable to induce protective antibodies. However, this protection istype-specific. Although capsular specific antibodies have been shown tobe highly protective, it remains unclear what concentration of theseserotype-specific antibodies protect against disease and more recentlyit has become clear that opsonic activity and avidity of theseantibodies are more critical determinants of protection thanconcentration.

The importance of surface proteins in human immunity to S. agalactiaealready has been appreciated. It is apparent that all serotypes expresssurface proteins with activity relevant to host immune defense. Thealpha C protein, beta C protein, Rib and Sip proteins arewell-characterized biochemically and genetically, and have also beenshown to immunogenic and protective in animal models ({Michel, J. etal., 1991}; {Brodeur, B. et al., 2000}; {Larsson, C. et al., 1999};{Cheng, Q. et al., 2002}). The major problem with these proteins asvaccine candidates seems to be their variability in prevalence among thedifferent clinical isolates of GBS. The Rib protein for example ispresent in serotype III GBS, but missing from type V, which responsiblefor significant portion of disease worldwide. Some other surfaceproteins are characterized as being immunogenic, but there is a limitedsystematic work done to identify most of the immunogenic proteins ofGBS.

Thus, there remains a need for an effective treatment to prevent orameliorate GBS infections. A vaccine could not only prevent infectionsby GBS, but more specifically prevent or ameliorate colonization of hosttissues (esp. in the birth canal), thereby reducing the incidence oftransmission from mother to fetus. Reducing the incidence of acuteinfection and carriage of the organism would lead to prevention ofinvasive diseases in newborns—pneumonia, bacteremia, meningitis, andsepsis. Vaccines capable of showing cross-protection against themajority of S. agalactiae strains causing human infections could also beuseful to prevent or ameliorate infections caused by all otherstreptococcal species, namely groups A, C and G.

A vaccine can contain a whole variety of different antigens. Examples ofantigens are whole-killed or attenuated organisms, subfractions of theseorganisms/tissues, proteins, or, in their most simple form, peptides.Antigens can also be recognized by the immune system in form ofglycosylated proteins or peptides and may also be or containpolysaccharides or lipids. Short peptides can be used since for examplecytotoxic T-cells (CTL) recognize antigens in form of short usually 8-11amino acids long peptides in conjunction with major histocompatibilitycomplex (MHC). B-cells can recognize linear epitopes as short as 4-5amino acids, as well as three-dimensional structures (conformationalepitopes). In order to obtain sustained, antigen-specific immuneresponses, adjuvants need to trigger immune cascades that involve allcells of the immune system. Primarily, adjuvants are acting, but are notrestricted in their mode of action, on so-called antigen presentingcells (APCs). These cells usually first encounter the antigen(s)followed by presentation of processed or unmodified antigen to immuneeffector cells. Intermediate cell types may also be involved. Onlyeffector cells with the appropriate specificity are activated in aproductive immune response. The adjuvant may also locally retainantigens and co-injected other factors. In addition the adjuvant may actas a chemoattractant for other immune cells or may act locally and/orsystemically as a stimulating agent for the immune system.

Vaccine development since the late 1970s has focused on the capsularpolysaccharides, but a safe, effective product is still not available.However, vaccine against S. agalactiae is ranked as one of the mostimportant for development and administration to infants and high-riskadults. Currently vaccines against this infection are only in theresearch stages of development. Efforts are focused on using capsularpolysaccharide (CPS) as immunogens, either with or without conjugationto protein {Paoletti, L. et al., 2002}. However, there are severalarguments against the use of polysaccharide-based vaccine.Polysaccharides induce IgG2 antibodies, which cross the placenta lessefficiently then IgG1 or IgG3 antibodies. It is especially a problem forthe most susceptible neonates, the still-borns since placental antibodytransfer is low before weeks 32-34. It is estimated that ˜10% ofdeliveries occur before the 34^(th) pregnancy week.

Protein conjugate vaccines are no doubt a great new addition to theamarmatorium in the battle against GBS disease, but the vaccine cancontain only a limited number of GBS serotypes and given adequateecological pressure, replacement disease by non-vaccine serotypesremains a real threat, particularly in areas with very high diseaseburden. Moreover, polysaccharide antigens used for active immunizationdo not provide immunological memory in humans. Conjugation of CPS tonon-GBS related immunogenic protein carriers (e.g. tetanus toxoid,cholera toxin B subunit, etc.) has been shown to beneficial in inducinghigher concentrations of antibodies in vaccinees, but it does notprovide pathogen-specific B cell and T cell epitopes which would recruitmemory B and T cells during a real infection to support the mosteffective host response. To be able to supplement the CPS vaccines withproteins fulfilling these criteria it is necessary to identify conservedimmunogenic GBS-specific surface proteins.

All these insufficiencies suggest that there is a need to develop newgeneration vaccines composed of proteins, or their derivatives,expressed by all strains under in vivo conditions with the ability toinduce opsonizing and/or neutralizing antibodies in humans.

There is a great potential for passive antibody-based therapy. Therehave been already attempts to use human intravenous immunoglobulin(IVIG) preparations for prevention. Recent advances in the technology ofmonoclonal antibody production provide the means to generate humanantibody reagents and reintroduce antibody therapies, while avoiding thetoxicities associated with serum therapy. Immunoglobulins are anextremely versatile class of antimicrobial proteins that can be used toprevent and treat emerging infectious diseases. Antibody therapy hasbeen effective against a variety of diverse microorganisms (reviewed in{Burnie, J. et al., 1998}). Anti-GBS mAbs could be given therapeuticallyto every newborn that develop invasive diseases or preventively to lowbirth-weight and premature neonates.

During the last decade the immunogenicity and protective capacity ofseveral GBS proteins have been described in animal models and these arenow being explored for the development of species-common protein basedvaccines. Such proteins are the GBS surface proteins Sip {Brodeur, B. etal., 2000}, rib,

-protein and {Michel, J. et al., 1991}.

Certain proteins or enzymes displayed on the surface of gram-positiveorganisms significantly contribute to pathogenesis, are involved in thedisease process caused by these pathogens. Often, these proteins areinvolved in direct interactions with host tissues or in concealing thebacterial surface from the host defense mechanisms {Navarre, W. et al.,1999}. S. agalactiae is not an exception in this regard. Several surfaceproteins are characterized as virulence factors, important for GBSpathogenicity ((reviewed in (Paoletti L. C. et al. in Gram positivepathogens, ed. by Fischetti V. A et al., ASM Press 2000, pp 137-153);{Paoletti, L. et al., 2002}). If antibodies to these proteins couldoffer better protection to humans then polysaccharides, they couldprovide the source of a novel, protein-based GBS vaccine to be used inconjunction with or in place of the more traditional capsularpolysaccharide vaccine. The use of some of the above-described proteinsas antigens for a potential vaccine as well as a number of additionalcandidates resulted mainly from a selection based on easiness ofidentification or chance of availability. There is a demand to identifyrelevant antigens for S. agalactiae in a more comprehensive way.

The present inventors have developed a method for identification,isolation and production of hyperimmune serum reactive antigens from aspecific pathogen, especially from Staphylococcus aureus andStaphylococcus epidermidis (WO 02/059148). However, given thedifferences in biological property, pathogenic function and geneticbackground, Streptococcus agalactiae is distinctive from Staphylococcusstrains. Importantly, the selection of sera for the identification ofantigens from S. agalactiae is different from that applied to the S.aureus screens. Four major types of human antibody sources werecollected for that purpose. First, healthy pregnant women who weretested negative for cervical and anorectal carriage of GBS. This donorgroup represents the most important source of antibodies. In addition totheir serum samples, human cervical secretions collected with cervicalwicks, containing secretory IgA (sIgA) were also used for antigenidentification and validation. The main value of this collection is thatsIgA can be considered the major immune effector molecule on mucosalsurfaces. Second, healthy pregnant women colonized with GBS whosenewborn remained GBS-free (although with antibiotic prevention). Third,adults below <45 years of age without clinical disease. Four, naïveindividuals, young children between 5 and 10 months of age, after theyalready lost maternal antibodies and have not acquired GBS-specific onesdue to the lack of GBS disease.

To be able to select for relevant serum sources, a series of ELISAs andimmunoblotting experiments measuring anti-S. agalactiae IgG and IgAantibody levels were performed with bacterial lysates and culturesupernatant proteins. Sera from high titer carriers and non-carrierswere included in the genomic-based antigen identification. This approachfor selection of human sera is basically very different from that usedfor S. aureus, where carriage or non-carriage state couldn't beassociated with antibody levels.

The present invention uses high throughput genomic method to identify invivo expressed pathogen-specific proteins with the ability to induceantibodies in humans during the course of infections and colonization.

The genomes of the two bacterial species S. agalactiae and S. aureus byitself show a number of important differences. The genome of S.agalactiae contains app. 2.2 Mb, while S. aureus harbors 2.85 Mb. Theyhave an average GC content of 35.7 and 33%, respectively andapproximately 30 to 45% of the encoded genes are not shared between thetwo pathogens. In addition, the two bacterial species require differentgrowth conditions and media for propagation. A list of the mostimportant diseases, which can be inflicted by the two pathogens ispresented below. S. aureus causes mainly nosocomial, opportunisticinfections: impetigo, folliculitis, abscesses, boils, infectedlacerations, endocarditis, meningitis, septic arthritis, pneumonia,osteomyelitis, scalded skin syndrome (SSS), toxic shock syndrome. S.agalactiae causes mainly neonatal infections and diseases in elderly,such as bacteremia, sepsis, wound infection, osteomyelitis andmeningitis.

The complete genome sequence of a capsular serotype III isolate of S.agalactiae, designated NEM316 (ATCC 12403) was determined by the randomshotgun sequencing strategy (GenBank accession number AL732656; seewww.tigr.org/tigrscripts/CMR2/CMRHomePage.spl). {Glaser, P. et al.,2002}.

The problem underlying the present invention was to provide means forthe development of medicaments such as vaccines against S. agalactiaeinfection. More particularly, the problem was to provide an efficient,relevant and comprehensive set of nucleic acid molecules or hyperimmuneserum reactive antigens from S. agalactiae that can be used for themanufacture of said medicaments.

Therefore, the present invention provides an isolated nucleic acidmolecule encoding a hyperimmune serum reactive antigen or a fragmentthereof comprising a nucleic acid sequence, which is selected from thegroup consisting of:

-   -   a) a nucleic acid molecule having at least 70% sequence identity        to a nucleic acid molecule selected from Seq ID No 14, 90,        157-216.    -   b) a nucleic acid molecule which is complementary to the nucleic        acid molecule of a),    -   c) a nucleic acid molecule comprising at least 15 sequential        bases of the nucleic acid molecule of a) or b)    -   d) a nucleic acid molecule which anneals under stringent        hybridisation conditions to the nucleic acid molecule of a), b),        or c)    -   e) a nucleic acid molecule which, but for the degeneracy of the        genetic code, would hybridize to the nucleic acid molecule        defined in a), b), c) or d).

According to a preferred embodiment of the present invention thesequence identity is at least 80%, preferably at least 95%, especially100%.

Furthermore, the present invention provides an isolated nucleic acidmolecule encoding a hyperimmune serum reactive antigen or a fragmentthereof comprising a nucleic acid sequence selected from the groupconsisting of

-   -   a) a nucleic acid molecule having 96% or more than 96%,        preferably at least 98%, especially 100% sequence identity to a        nucleic acid molecule selected from Seq ID No 1, 3, 5-13, 15,        18-25, 27-31, 33-36, 39-68, 70-85, 92-100, 103-126, 128-145,        147, 149-156, 217, 435-448 and 463-474.    -   b) a nucleic acid molecule which is complementary to the nucleic        acid molecule of a),    -   c) a nucleic acid molecule comprising at least 15 sequential        bases of the nucleic acid molecule of a) or b)    -   d) a nucleic acid molecule which anneals under stringent        hybridisation conditions to the nucleic acid molecule of a), b)        or c),    -   e) a nucleic acid molecule which, but for the degeneracy of the        genetic code, would hybridize to the nucleic acid defined in a),        b), c) or d).

According to another aspect, the present invention provides an isolatednucleic acid molecule encoding a hyperimmune serum reactive antigen or afragment thereof comprising a nucleic acid sequence selected from thegroup consisting of

-   -   a) a nucleic acid molecule having 98% or more than 98%,        especially 100% sequence identity to a nucleic acid molecule        selected from Seq ID No 32, 86, 91, 101, 127.    -   b) a nucleic acid molecule which is complementary to the nucleic        acid of a),    -   c) a nucleic acid molecule which, but for the degeneracy of the        genetic code, would hybridize to the nucleic acid defined in a),        b), c) or d).

Preferably, the nucleic acid molecule is DNA or RNA.

According to a preferred embodiment of the present invention, thenucleic acid molecule is isolated from a genomic DNA, especially from aS. agalactiae genomic DNA.

According to the present invention a vector comprising a nucleic acidmolecule according to any of the present invention is provided.

In a preferred embodiment the vector is adapted for recombinantexpression of the hyperimmune serum reactive antigens or fragmentsthereof encoded by the nucleic acid molecule according to the presentinvention.

The present invention also provides a host cell comprising the vectoraccording to the present invention. According to another aspect thepresent invention further provides a hyperimmune serum-reactive antigencomprising an amino acid sequence being encoded by a nucleic acidmolecule according to the present invention.

In a preferred embodiment the amino acid sequence (polypeptide) isselected from the group consisting of Seq ID No 231, 307, 374-433.

In another preferred embodiment the amino acid sequence (polypeptide) isselected from the group consisting of Seq ID No 218, 220, 222-230, 232,235-242, 244-248, 250-253, 256-285, 287-302, 309-317, 320-343, 345-362,364, 366-373, 434, 449-462 and 475-486.

In a further preferred embodiment the amino acid sequence (polypeptide)is selected from the group consisting of Seq ID No 249, 303, 308, 318,344.

According to a further aspect the present invention provides fragmentsof hyperimmune serum-reactive antigens selected from the groupconsisting of peptides comprising amino acid sequences of column“predicted immunogenic aa” and “location of identified immunogenicregion” of Table 1A, especially peptides comprising amino acid 4-20,35-44, 65-70, 73-87, 92-98, 112-137, 152-161, 177-186, 193-200, 206-213,229-255, 282-294, 308-313, 320-326, 349-355, 373-384, 388-406, 420-425and 115-199 of Seq ID No 218; 5-24, 35-41, 44-70, 73-89, 103-109,127-143, 155-161, 185-190, 192-207, 212-219, 246-262, 304-336, 372-382,384-393, 398-407, 412-418, 438-444, 1-75, 76-161 and 164-239 of Seq IDNo 219; 4-10, 16-58, 60-71, 77-92, 100-126, 132-146, 149-164, 166-172,190-209, 214-220, 223-229, 241-256, 297-312, 314-319, 337-343, 351-359,378-387, 398-418, 421-428, 430-437, 440-448, 462-471, 510-519, 525-536,552-559, 561-568, 573-582, 596-602, 608-630, 637-649, 651-665, 681-702,714-732, 739-745, 757-778, 790-805, 807-815, 821-829, 836-842, 846-873,880-903, 908-914, 916-923, 931-940, 943-948, 956-970, 975-986, 996-1015,1031-1040, 1051-1069, 1072-1095, 1114-1119, 1130-1148, 1150-1157,1169-1176, 1229-1238 and 802-812 of Seq ID No 220; 5-12, 14-26, 35-47,52-67, 72-78, 83-98, 121-141, 152-159, 163-183, 186-207, 209-257,264-277, 282-299, 301-309, 312-318, 324-339, 358-368, 372-378, 387-397,425-431 and 46-291 of Seq ID No 221; 29-38, 44-64, 70-76, 78-87, 94-100,102-112, 119-134, 140-149, 163-173, 178-186, 188-194, 207-234, 247-262,269-290 and 73-92 of Seq ID No 222; 10-28, 36-63, 77-87, 103-119,127-136, 141-169, 171-183, 195-200, 207-232, 236-246, 251-265, 268-283,287-297, 314-322, 335-343, 354-363, 384-390, 405-411, 419-436, 443-455,467-473, 480-513, 518-529, 550-557, 565-585, 602-608, 616-625, 632-660,665-677, 685-701, 726-736, 738-747, 752-761, 785-796, 801-813, 838-853,866-871 and 757-774 of Seq ID No 223; 31-38, 61-66, 74-81, 90-115,123-145, 154-167, 169-179, 182-193, 200-206, 238-244, 267-272 and235-251 of Seq ID No 224; 19-25, 38-54, 56-64, 66-72, 74-92, 94-100,116-129, 143-149, 156-183, 204-232, 253-266, 269-275, 294-307 and241-313 of Seq ID No 225; 5-34, 50-56, 60-65, 74-85, 89-97, 108-119,159-165, 181-199, 209-225, 230-240, 245-251, 257-262, 274-282, 300-305and 64-75 of Seq ID No 226; 5-13, 16-21, 27-42, 45-52, 58-66, 74-87,108-114, 119-131 and 39-51 of Seq ID No 227; 6-23, 46-54, 59-65, 78-84,100-120, 128-133, 140-146, 159-165, 171-183, 190-204, 224-232, 240-248,250-259, 274-280, 288-296, 306-315 and 267-274 of Seq ID No 228; 5-12,15-24, 26-36, 42-65, 68-80, 82-104, 111-116, 125-144, 159-167, 184-189,209-218, 235-243, 254-265, 269-283, 287-300, 306-316, 318-336, 338-352,374-392 and 162-174 of Seq ID No 229; 30-42, 45-54 and 25-37 of Seq IDNo 230; 10-30, 53-59, 86-95, 116-130, 132-147, 169-189, 195-201,212-221, 247-256, 258-265, 278-283, 291-298, 310-316, 329-339, 341-352,360-367, 388-396, 398-411, 416-432, 443-452, 460-466, 506-512, 515-521,542-548 and 419-431 of Seq ID No 231; 4-27, 30-53, 60-67, 70-90, 92-151,159-185, 189-195, 198-210, 215-239 and 173-189 of Seq ID No 232; 4-26,41-54, 71-78, 116-127, 140-149, 151-158, 161-175, 190-196, 201-208,220-226, 240-252, 266-281, 298-305, 308-318, 321-329, 344-353, 372-378,384-405, 418-426, 429-442, 457-463, 494-505, 514-522 and 174-188 of SeqID No 233; 17-25, 27-39, 61-67, 81-89, 99-110, 120-131, 133-139,147-161, 167-172, 179-185, 192-198, 203-213, 226-238, 243-258, 261-267,284-290, 296-307, 311-328, 340-352, 356-371 and 239-256 of Seq ID No234; 8-30, 40-49, 67-80, 114-123, 126-142, 152-162, 188-194 and 57-70 ofSeq ID No 235; 4-23, 28-34, 36-47, 50-61, 76-81, 89-94, 96-104, 112-119,126-146, 155-181, 195-200, 208-214, 220-229, 244-260, 263-276, 282-288,292-300, 317-323, 336-351, 353-359, 363-375, 382-399, 415-432, 444-455,458-471, 476-481, 484-492, 499-517, 522-529, 535-541, 543-568, 572-584,586-600, 607-617, 626-637, 656-675 and 282-297 of Seq ID No 236; 6-24,30-35, 38-45, 63-91, 134-140, 146-160, 167-188, 214-220, 226-234,244-250, 260-270, 286-301, 316-329, 340-371, 429-446, 448-459, 474-481,485-491, 512-526, 537-544, 550-565, 573-583, 596-613, 621-630, 652-658and 87-97 of Seq ID No 237; 8-20, 26-48, 56-67, 76-86, 94-109, 115-121,123-129, 143-160, 178-186, 191-198, 201-208, 221-236, 238-244, 260-268and 237-247 of Seq ID No 238; 4-40, 42-57, 73-87, 98-117, 126-135,150-156, 166-174, 196-217, 231-236, 248-258, 276-284, 293-301, 307-313,339-347, 359-365, 375-387, 395-402, 428-440, 445-456, 485-490, 497-505,535-541, 547-555, 610-625, 648-656, 665-671 and 448-528 of Seq ID No239; 10-18, 39-45, 51-61, 80-96, 98-106, 110-115, 158-172, 174-183,191-200, 220-237, 249-255, 274-289, 308-324, 331-341, 372-381, 384-397,405-414 and 322-338 of Seq ID No 240; 30-36, 38-56, 85-108, 134-147,149-160, 163-183, 188-201, 206-211, 219-238, 247-254 and 5-13 of Seq IDNo 241; 11-40, 98-103, 110-115, 133-145, 151-159, 172-179, 192-201,204-212, 222-228, 235-245, 258-268, 283-296, 298-309, 322-329, 342-351,354-362, 372-378, 385-393, 407-418, 495-516 and 1-148 of Seq ID No 242;5-19, 21-36, 73-94, 112-119, 122-137, 139-145, 152-167, 184-190,198-204, 208-224, 249-265, 267-281, 299-304, 309-317, 326-333, 356-364,368-374, 381-389, 391-414, 419-425, 430-435 and 113-140 of Seq ID No243; 45-54, 59-67, 78-91 and 15-23 of Seq ID No 244; 11-22, 33-47,52-80, 88-112, 124-129 and 6-25 of Seq ID No 245; 26-41, 51-63, 80-89,93-115, 150-163, 187-193, 220-237, 240-249, 286-294, 296-306, 316-329,345-353, 361-370, 407-425, 428-437, 474-482, 484-494, 504-517, 533-541,549-558, 595-613, 616-625, 660-668, 673-685, 711-726, 736-744, 749-761,787-802, 812-820, 825-837, 863-878, 888-896, 901-913, 939-954, 964-972,977-989, 1003-1008, 1016-1022, 1028-1034, 1041-1053, 1059-1074,1101-1122, 420-511 and 581-704 of Seq ID No 246; 18-25, 27-55, 71-83,89-95, 102-113, 120-146, 150-156, 174-185 and 159-175 of Seq ID No 247;24-30, 38-56, 63-68, 87-93, 136-142, 153-164, 183-199, 213-219, 226-234,244-261, 269-278, 283-289, 291-297, 320-328, 330-336, 340-346, 348-356,358-366, 382-387, 401-408, 414-419, 449-455, 468-491, 504-512, 531-537,554-560, 597-608, 621-627, 632-643, 650-662, 667-692, 703-716, 724-737,743-758, 783-794, 800-818, 846-856 and 806-884 of Seq ID No 248; 4-14,21-39, 86-92, 99-107, 121-131, 136-144, 147-154, 158-166, 176-185,193-199, 207-222, 224-230 and 117-136 of Seq ID No 249; 65-76, 85-97,103-109, 115-121, 125-146, 163-169, 196-205, 212-219, 228-237, 241-247,254-262, 269-288, 294-303, 305-313, 328-367, 395-401, 405-412, 418-429,437-447, 481-488, 506-513, 519-524, 530-541, 546-557 and 266-284 of SeqID No 250; 5-14, 37-42, 49-71, 78-92, 97-112, 127-136, 147-154, 156-163,186-198, 216-225, 233-243, 248-253, 295-307, 323-332, 359-366, 368-374,380-398 and 194-223 of Seq ID No 251; 4-11, 33-39, 45-72, 100-113,119-129, 136-144, 169-175, 177-185, 200-208, 210-219, 262-276, 278-297,320-326, 336-344, 347-362, 381-394, 443-453 and 438-454 of Seq ID No252; 4-29, 31-52, 55-61, 95-110, 138-158, 162-171, 179-187, 202-229,239-248, 251-256, 262-267, 269-285, 304-310, 351-360, 362-368, 381-388,415-428, 435-440, 448-458 and 161-178 of Seq ID No 253; 4-17, 19-28,32-43, 47-59, 89-110, 112-126, 128-134, 140-148, 152-161, 169-184,191-204, 230-235, 255-264, 328-338, 341-347, 401-409, 413-419, 433-441,449-458, 463-468, 476-482, 486-492, 500-506, 529-545 and 305-381 of SeqID No 254; 10-29, 38-45, 53-61, 134-145, 152-160, 163-170, 202-208,219-229, 248-258, 266-275, 282-288, 315-320, 328-334, 377-385, 392-402,418-424, 447-453, 460-471, 479-487, 491-497, 500-507, 531-537, 581-594,615-623, 629-635, 644-652, 659-666, 668-678, 710-717, 719-728, 736-741,747-760, 766-773, 784-789, 794-800, 805-817, 855-861, 866-887 and698-715 of Seq ID No 255; 16-26, 29-37, 44-58, 62-68, 74-80, 88-95,97-120, 125-144, 165-196 and 58-72 of Seq ID No 256; 14-21, 23-46,49-60, 63-74, 78-92, 96-103, 117-129, 134-161, 169-211, 217-231,239-248, 252-281, 292-299, 313-343 and 243-257 of Seq ID No 257; 11-27,46-52, 67-72, 76-84, 91-112, 116-153, 160-175, 187-196, 202-211, 213-220and 43-76 of Seq ID No 258; 5-29, 37-56, 78-86, 108-118, 152-161 and120-130 of Seq ID No 259; 8-14, 19-41, 52-66, 75-82, 87-92, 106-121,127-133, 136-143, 158-175, 180-187, 196-204, 221-228, 239-245, 259-265,291-306, 318-323, 328-340, 352-358, 361-368, 375-381, 391-399, 411-418,431-442, 446-455, 484-496, 498-510, 527-533, 541-549, 558-565, 575-585,587-594, 644-655, 661-668, 671-677 and 184-196 of Seq ID No 260; 4-22,29-38, 55-62, 75-81, 102-107, 110-134, 143-150, 161-167, 172-179,191-215, 223-233, 241-247, 251-264, 266-272, 288-309, 340-352, 354-366,394-402, 414-438 and 198-218 of Seq ID No 261; 24-44, 49-70, 80-91,105-118, 128-136, 140-154 and 77-92 of Seq ID No 262; 5-22, 31-36,41-47, 67-74, 83-90, 105-122, 135-143, 160-167 and 118-129 of Seq ID No263; 4-25, 33-73, 81-93, 96-106, 114-120, 122-128, 130-172, 179-208,210-241, 251-283, 296-301 and 92-100 of Seq ID No 264; 14-24, 29-38,43-50, 52-72, 86-97, 101-107, 110-125, 127-141, 145-157, 168-175,177-184, 186-195, 205-226, 238-250, 255-261, 284-290, 293-304, 307-314,316-323, 325-356, 363-371, 383-390, 405-415, 423-432, 442-454, 466-485,502-511, 519-527, 535-556, 558-565, 569-574, 612-634, 641-655, 672-686,698-709, 715-722, 724-732, 743-753, 760-769, 783-792, 818-825, 830-839,842-849, 884-896, 905-918, 926-940, 957-969, 979-1007, 1015-1021,1049-1057 and 336-349 of Seq ID No 265; 6-16, 26-31, 33-39, 62-73,75-85, 87-100, 113-123, 127-152, 157-164, 168-181, 191-198, 208-214,219-226, 233-254, 259-266, 286-329 and 181-195 of Seq ID No 266; 4-13,32-39, 53-76, 99-108, 110-116, 124-135, 137-146, 149-157, 162-174,182-190, 207-231, 242-253, 255-264, 274-283, 291-323, 334-345, 351-360,375-388, 418-425, 456-474, 486-492, 508-517, 520-536, 547-560, 562-577,31-45 and 419-443 of Seq ID No 267; 15-26, 30-37, 42-49, 58-90, 93-99,128-134, 147-154, 174-179, 190-197, 199-205, 221-230, 262-274, 277-287,300-314, 327-333, 343-351, 359-377, 388-396, 408-413, 416-425, 431-446and 246-256 of Seq ID No 268; 5-26, 34-42, 47-54, 61-67, 71-104,107-115, 131-138, 144-153, 157-189, 196-202, 204-210, 228-245, 288-309,316-329, 332-341, 379-386, 393-399, 404-412, 414-421, 457-468, 483-489,500-506, 508-517, 523-534, 543-557, 565-580, 587-605, 609-617, 619-627,631-636, 640-646, 662-668, 675-682, 705-710, 716-723, 727-732, 750-758,784-789, 795-809, 869-874, 14-138, 166-286, 372-503, 674-696 and 754-859of Seq ID No 269; 5-17, 32-38, 40-47, 80-89, 113-119, 125-137, 140-154,157-163, 170-177, 185-199, 213-225, 228-236, 242-248, 277-290, 292-305,323-333, 347-353, 364-370, 385-394, 399-406, 423-433, 441-451, 462-474,477-487 and 116-124 of Seq ID No 270; 7-16, 18-30, 32-49, 53-61, 63-85,95-101, 105-115, 119-134, 143-150, 159-178, 185-202, 212-229, 236-250,254-265, 268-294 and 63-72 of Seq ID No 271; 4-12, 19-47, 73-81, 97-103,153-169, 188-198, 207-213, 217-223, 236-242, 255-265, 270-278, 298-305,309-317, 335-347, 354-363, 373-394, 419-424, 442-465, 486-492, 500-507,542-549, 551-558, 560-572, 580-589, 607-614, 617-623, 647-653, 666-676,694-704, 706-714, 748-754, 765-772, 786-792, 795-806 and 358-370 of SeqID No 272; 18-28, 30-38, 40-46, 49-55, 69-78, 82-98, 104-134, 147-153,180-190, 196-202, 218-236, 244-261, 266-273, 275-286, 290-295, 301-314,378-387, 390-395, 427-434 and 290-305 of Seq ID No 273; 4-13, 20-31,39-51, 54-61, 69-84, 87-105, 117-124 and 108-125 of Seq ID No 274;24-34, 43-54, 56-66, 68-79 and 50-69 of Seq ID No 275; 5-43, 71-77,102-131, 141-148, 150-156, 159-186, 191-207, 209-234, 255-268, 280-286,293-299, 317-323, 350-357, 363-372, 391-397, 406-418, 428-435, 455-465,484-497, 499-505, 525-531, 575-582, 593-607, 621-633, 638-649, 655-673,684-698, 711-725, 736-741, 743-752, 759-769, 781-793, 813-831, 843-853,894-905, 908-916, 929-946, 953-963, 970-978, 1001-1007, 1011-1033,165-178 and 818-974 of Seq ID No 276; 16-44, 63-86, 98-108, 185-191,222-237, 261-274, 282-294, 335-345, 349-362, 374-384, 409-420, 424-430,440-447, 453-460, 465-473, 475-504, 522-534, 538-551, 554-560, 567-582,598-607, 611-619, 627-640, 643-653, 655-661, 669-680, 684-690, 701-707,715-731, 744-750, 756-763, 768-804, 829-837, 845-853, 855-879, 884-890,910-928, 77-90, 144-212, 279-355, 434-536, 782-810 and 875-902 of Seq IDNo 277; 4-22, 29-41, 45-51, 53-66, 70-77, 86-95, 98-104, 106-124,129-135, 142-151, 153-161, 169-176, 228-251, 284-299, 331-337, 339-370,380-387, 393-398, 406-411, 423-433, 440-452, 461-469, 488-498, 501-516,523-530, 532-559, 562-567, 570-602, 612-628, 630-645, 649-659, 666-672,677-696, 714-723, 727-747 and 212-227 of Seq ID No 278; 4-9, 17-31,35-41, 56-61, 66-75, 81-87, 90-124, 133-138, 149-163, 173-192, 213-219,221-262, 265-275, 277-282, 292-298, 301-307, 333-346, 353-363, 371-378,419-430, 435-448, 456-469, 551-570, 583-599, 603-612 and 275-291 of SeqID No 279; 28-34, 53-58, 72-81, 100-128, 145-154, 159-168, 172-189,217-225, 227-249, 256-263, 299-309, 322-330, 361-379, 381-388, 392-401,404-417, 425-436, 440-446, 451-464, 469-487, 502-511, 543-551, 559-564,595-601, 606-612, 615-626, 633-642, 644-650, 664-670, 674-684, 692-701,715-723, 726-734, 749-756, 763-771, 781-787, 810-843, 860-869, 882-889,907-917, 931-936, 941-948, 951-958, 964-971, 976-993, 1039-1049,1051-1065, 1092-1121, 1126-1132, 1145-1151, 1158-1173, 1181-1192,1194-1208, 1218-1223, 1229-1243, 1249-1254, 1265-1279, 1287-1297,1303-1320, 1334-1341, 1343-1358, 1372-1382, 1406-1417, 1419-1425,1428-1434, 1441-1448, 1460-1473, 1494-1504, 1509-1514, 1529-1550,654-669 and 1400-1483 of Seq ID No 280; 10-16, 20-25, 58-65, 97-109,118-132, 134-146, 148-155, 186-195, 226-233, 244-262, 275-284, 295-310,317-322, 330-339, 345-351, 366-375, 392-403, 408-415, 423-430, 435-444,446-457, 467-479, 486-499, 503-510, 525-537, 540-585, 602-612, 614-623,625-634, 639-645, 650-669, 700-707, 717-724, 727-739, 205-230 and733-754 of Seq ID No 281; 5-22, 37-43, 72-81, 105-113, 128-133, 148-160,188-194, 204-230, 238-245, 251-257 and 194-213 of Seq ID No 282; 16-21,35-41, 56-72, 74-92, 103-109 and 62-68 of Seq ID No 283; 4-15, 17-82,90-104, 107-159, 163-170, 188-221, 234-245, 252-265 and 220-235 of SeqID No 284; 16-22, 36-46, 61-75, 92-107, 113-121, 139-145, 148-160 and30-42 of Seq ID No 285; 4-12, 20-26, 43-49, 55-62, 66-78, 121-127,135-141, 146-161, 164-170, 178-189, 196-205, 233-238, 269-279, 288-318,325-332, 381-386, 400-407 and 328-346 of Seq ID No 286; 5-12, 31-49,57-63, 69-79, 89-97, 99-114, 116-127, 134-142, 147-154, 160-173,185-193, 199-204, 211-222, 229-236, 243-249, 256-274 and 58-68 of Seq IDNo 287; 10-20, 28-34, 39-53, 68-79, 84-90, 99-106 and 73-79 of Seq ID No288; 14-37, 45-50, 61-66, 77-82, 93-98, 109-114, 125-130, 141-146,157-162, 173-178, 189-194, 205-210, 221-226, 237-242, 253-258, 269-274,285-290, 301-306, 316-332, 349-359, 371-378, 385-406, 34-307 and 312-385of Seq ID No 289; 4-10, 17-38, 50-85, 93-99, 109-116, 128-185, 189-197,199-210, 223-256, 263-287, 289-312, 327-337, 371-386, 389-394, 406-419,424-432, 438-450, 458-463, 475-502, 507-513, 519-526, 535-542, 550-567and 361-376 of Seq ID No 290; 10-39, 42-93, 100-144, 155-176, 178-224,230-244, 246-255, 273-282, 292-301, 308-325, 332-351, 356-361, 368-379,386-393, 400-421 and 138-155 of Seq ID No 291; 5-11, 17-34, 40-45,50-55, 72-80, 101-123, 145-151, 164-172, 182-187, 189-195, 208-218,220-241, 243-252, 255-270, 325-331, 365-371, 391-398, 402-418, 422-428,430-435, 443-452, 463-469, 476-484, 486-494, 503-509, 529-553, 560-565,570-590, 608-614, 619-627, 654-661, 744-750, 772-780, 784-790, 806-816,836-853, 876-885, 912-918, 926-933, 961-975, 980-987, 996-1006,1016-1028, 1043-1053, 1057-1062, 994-1003 and 1033-1056 of Seq ID No292; 17-45, 64-71, 73-81, 99-109, 186-192, 223-238, 262-275, 283-295,336-346, 350-363, 375-385, 410-421, 425-431, 441-448, 454-463, 468-474,476-512, 523-537, 539-552, 568-583, 599-608, 612-620, 628-641, 644-654,656-662, 670-681, 685-695, 702-708, 716-723, 725-735, 757-764, 769-798,800-806, 808-816, 826-840, 846-854, 856-862, 874-881, 885-902, 907-928,274-350 and 443-513 of Seq ID No 293; 4-22, 29-41, 45-51, 53-61, 70-76,85-92, 99-104, 111-122, 134-140, 142-154, 163-174, 224-232, 255-265,273-279, 283-297, 330-335, 337-348, 356-367, 373-385, 391-396, 421-431,442-455, 475-485, 493-505, 526-538, 544-561, 587-599, 605-620, 622-651,662-670, 675-681, 687-692, 697-712, 714-735 and 252-262 of Seq ID No294; 4-12, 15-35, 40-46, 50-59, 67-94, 110-128, 143-169, 182-188,207-215, 218-228, 238-250 and 74-90 of Seq ID No 295; 9-18, 42-58,78-85, 88-95, 97-106, 115-122, 128-134, 140-145, 154-181, 186-202,204-223, 261-267, 269-278, 284-293, 300-336, 358-368 and 12-29 of Seq IDNo 296; 7-34, 46-53, 62-72, 82-88, 100-105, 111-117, 132-137, 144-160,166-180, 183-189, 209-221, 231-236, 246-253, 268-282, 286-293, 323-336,364-372, 378-392, 422-433 and 388-405 of Seq ID No 297; 21-27, 34-50,72-77, 80-95, 164-177, 192-198, 202-220, 226-236, 239-247, 270-279,285-292, 315-320, 327-334, 348-355, 364-371, 388-397, 453-476, 488-497,534-545, 556-576, 582-588, 601-607, 609-616, 642-662, 674-681, 687-697,709-715, 721-727, 741-755 and 621-739 of Seq ID No 298; 4-14, 16-77,79-109 and 25-99 of Seq ID No 299; 4-9, 17-23, 30-37, 44-55, 65-72,77-93, 102-121, 123-132, 146-153 and 17-29 of Seq ID No 300; 4-18,25-41, 52-60, 83-92, 104-112, 117-123, 149-155, 159-167, 170-192,201-210, 220-227, 245-250 and 124-137 of Seq ID No 301; 8-25, 50-55,89-95, 138-143, 148-153, 159-169, 173-179, 223-238, 262-268, 288-295,297-308, 325-335, 403-409, 411-417, 432-446, 463-475, 492-501, 524-530,542-548, 561-574, 576-593, 604-609, 612-622, 637-654, 665-672, 678-685,720-725, 731-739, 762-767, 777-783, 820-838, 851-865, 901-908, 913-920,958-970, 1000-1006, 1009-1015, 1020-1026, 1043-1052, 1055-1061, 1-128,252-341, 771-793 and 1043-1058 of Seq ID No 302; 16-26, 33-46 and 64-76of Seq ID No 303; 4-27, 69-77, 79-101, 117-123, 126-142, 155-161,171-186, 200-206, 213-231, 233-244, 267-273, 313-329, 335-344, 347-370,374-379, 399-408, 422-443, 445-453, 461-468, 476-482, 518-534, 544-553,556-567, 578-595, 601-620, 626-636, 646-658, 666-681, 715-721, 762-768,778-785, 789-803, 809-819, 22-108, 153-318, 391-527 and 638-757 of SeqID No 304; 6-21, 32-43, 62-92, 104-123, 135-141, 145-152, 199-216,218-226, 237-247, 260-269, 274-283, 297-303, 1-72 and 127-211 of Seq IDNo 305; 6-26, 50-56, 83-89, 108-114, 123-131, 172-181, 194-200, 221-238,241-247, 251-259, 263-271, 284-292, 304-319, 321-335, 353-358, 384-391,408-417, 424-430, 442-448, 459-466, 487-500, 514-528, 541-556, 572-578,595-601, 605-613, 620-631, 635-648, 660-670, 673-679, 686-693, 702-708,716-725, 730-735, 749-755, 770-777, 805-811, 831-837, 843-851, 854-860,863-869, 895-901, 904-914, 922-929, 933-938, 947-952, 956-963,1000-1005, 1008-1014, 1021-1030, 1097-1103, 1120-1130, 1132-1140, 1-213,269-592 and 992-1120 of Seq ID No 306; 9-16, 33-39, 47-59, 65-79, 81-95,103-108, 115-123, 138-148, 163-171, 176-185, 191-196, 205-211, 213-221,224-256, 261-276, 294-302, 357-363, 384-390, 95-111 and 161-189 of SeqID No 307; 21-27, 35-45, 70-76, 92-105, 129-143, 145-155, 161-166,170-191, 204-211, 214-231, 234-246, 249-255, 259-275 and 1-18 of Seq IDNo 308; 21-35, 45-53, 56-64, 69-97 and 1-16 of Seq ID No 309; 25-33,41-47, 61-68, 86-101, 106-114, 116-129, 134-142, 144-156, 163-176,181-190, 228-251, 255-261, 276-292, 295-305, 334-357, 368-380, 395-410,424-429, 454-460, 469-482, 510-516, 518-527, 531-546, 558-570, 579-606,628-636, 638-645, 651-656, 668-674, 691-698, 717-734, 742-754, 765-770,792-797, 827-835, 847-859, 874-881, 903-909, 926-933, 942-961, 964-977,989-1004, 1010-1028, 1031-1047, 1057-1075, 1081-1095, 1108-1117,1138-1144, 1182-1189, 1193-1206, 1220-1229, 1239-1246, 1257-1267,1271-1279, 1284-1301, 1312-1320, 1329-1335, 1341-1347, 1358-1371,1399-1404, 1417-1426, 1458-1463, 1468-1476, 1478-1485, 1493-1506,1535-1541, 1559-1574, 1583-1590, 1595-1601, 1603-1611, 1622-1628,1634-1644, 1671-1685, 1689-1696, 1715-1720, 1734-1746, 1766-1775,1801-1806, 1838-1844, 1858-1871, 1910-1917, 1948-1955, 1960-1974,2000-2015, 2019-2036, 2041-2063, 748-847 and 1381-1391 of Seq ID No 310;5-12, 18-24, 27-53, 56-63, 96-113, 119-124, 131-136, 157-163, 203-209,215-223, 233-246, 264-273, 311-316, 380-389, 393-399, 425-433, 445-450,457-462, 464-470, 475-482, 507-513, 527-535, 542-548, 550-565, 591-602,607-613, 627-642, 644-664, 673-712, 714-732, 739-764, 769-782, 812-818,826-838, 848-854, 860-871, 892-906, 930-938, 940-954, 957-973, 990-998,1002-1021, 1024-1033, 1037-1042, 1050-1060, 1077-1083, 1085-1092,1100-1129, 1144-1161, 1169-1175, 1178-1189, 1192-1198, 1201-1207,1211-1221, 1229-1239, 1250-1270, 1278-1292, 1294-1300, 1314-1335,1344-1352, 1360-1374, 1394-1405, 1407-1414, 1416-1424, 1432-1452,1456-1462, 1474-1497, 1500-1510, 1516-1522, 1534-1542, 1550-1559,1584-1603, 1608-1627, 187-273 and 306-441 of Seq ID No 311; 70-80,90-97, 118-125, 128-140, 142-148, 154-162, 189-202, 214-222, 224-232,254-260, 275-313, 317-332, 355-360, 392-398, 425-432, 448-456, 464-470,476-482, 491-505, 521-528, 533-546, 560-567, 592-597, 605-614, 618-626,637-644, 646-653, 660-666, 677-691 and 207-227 of Seq ID No 312; 5-19,26-34, 37-55, 57-66, 69-83, 86-102, 115-134, 138-143, 154-172, 178-195,209-246, 251-257, 290-302, 306-311 and 256-266 of Seq ID No 313; 10-20,22-28, 35-57, 72-79, 87-103, 108-128, 130-144, 158-171, 190-198,225-242, 274-291, 301-315, 317-324, 374-385 and 353-365 of Seq ID No314; 4-9, 17-30, 34-54, 59-66, 73-94, 118-130, 135-150, 158-171,189-198, 219-239, 269-275, 283-301, 89-106 and 176-193 of Seq ID No 315;14-20, 22-74, 77-86, 89-99, 104-109, 126-135, 154-165, 181-195, 197-212,216-224, 264-275 and 107-118 of Seq ID No 316; 4-18, 21-38, 63-72,101-109, 156-162, 165-179, 183-192, 195-210, 212-218, 230-239, 241-256,278-290, 299-311, 313-322, 332-341, 348-366, 386-401, 420-426, 435-450,455-460, 468-479, 491-498, 510-518, 532-538, 545-552, 557-563, 567-573,586-595, 599-609, 620-626, 628-636, 652-657, 665-681 and 1-198 of Seq IDNo 317; 4-10, 16-38, 51-68, 73-79, 94-115, 120-125, 132-178, 201-208,216-223, 238-266, 269-295, 297-304, 337-342, 347-356, 374-401, 403-422,440-447, 478-504, 510-516, 519-530, 537-544 and 191-206 of Seq ID No318; 12-40, 42-48, 66-71, 77-86, 95-102, 113-120, 129-137, 141-148,155-174, 208-214, 218-225, 234-240, 256-267, 275-283, 300-306, 313-321,343-350, 359-367, 370-383, 398-405, 432-439, 443-461, 492-508, 516-526,528-535 and 370-478 of Seq ID No 319; 6-14, 20-37, 56-62, 90-95, 97-113,118-125, 140-145, 161-170, 183-202, 237-244, 275-284, 286-305, 309-316,333-359, 373-401, 405-412 and 176-187 of Seq ID No 320; 33-44, 50-55,59-80, 86-101, 129-139, 147-153, 157-163, 171-176, 189-201, 203-224,239-245, 257-262, 281-287, 290-297, 304-320, 322-331, 334-350, 372-390,396-401, 71-88 and 353-372 of Seq ID No 321; 5-11, 15-24, 26-33, 40-47,75-88, 95-103, 105-112 and 17-30 of Seq ID No 322; 5-11, 16-39, 46-54,62-82, 100-107, 111-124, 126-150, 154-165, 167-183, 204-238, 245-295,301-313, 316-335 and 8-16 of Seq ID No 323; 4-19, 34-48, 69-74, 79-107,115-127, 129-135, 143-153, 160-169, 171-182 and 142-153 of Seq ID No324; 4-30, 65-74, 82-106, 110-120, 124-132, 135-140, 146-175, 179-184,190-196, 217-223, 228-233, 250-267, 275-292, 303-315, 322-332 and174-186 of Seq ID No 325; 9-16, 29-41, 47-57, 68-84, 87-109, 113-119,162-180, 186-193, 195-201, 203-208, 218-230, 234-243, 265-271, 281-292,305-312, 323-332, 341-347, 349-363, 368-374, 383-390, 396-410, 434-440,446-452, 455-464, 466-473, 515-522, 529-542, 565-570, 589-600, 602-613,618-623, 637-644, 1019-1027, 1238-1244, 1258-1264, 1268-1276, 1281-1292,1296-1302 and 883-936 of Seq ID No 326; 10-17, 23-32, 39-44, 54-72,75-81, 88-111, 138-154, 160-167, 178-185, 201-210, 236-252, 327-334,336-342, 366-376, 388-400, 410-430, 472-482, 493-526, 552-558, 586-592,598-603, 612-621, 630-635, 641-660 and 384-393 of Seq ID No 327; 4-22,24-39, 50-59, 73-84, 100-105, 111-117, 130-138, 155-161, 173-178,182-189, 205-215, 266-284, 308-313, 321-328, 330-337, 346-363, 368-374,388-395, 397-405, 426-434, 453-459, 482-492, 501-507, 509-515, 518-523,527-544, 559-590, 598-612, 614-629, 646-659, 663-684, 686-694, 698-721and 445-461 of Seq ID No 328; 14-22, 27-33 and 3-17 of Seq ID No 329;29-41, 66-73, 81-87, 90-108, 140-146, 150-159, 165-184, 186-196,216-226, 230-238, 247-253, 261-269 and 126-140 of Seq ID No 330; 5-12,16-25, 27-33, 36-45, 60-68, 83-88, 103-126 and 86-101 of Seq ID No 331;14-23, 36-47, 56-66, 84-89, 94-105, 111-127, 140-153, 160-174, 176-183,189-203, 219-225, 231-237, 250-257 and 194-227 of Seq ID No 332; 4-25,54-60, 64-71, 73-82, 89-106, 117-124, 157-169, 183-188, 199-210,221-232, 236-244, 255-264 and 58-98 of Seq ID No 333; 13-19, 26-36,41-53, 55-71, 77-84, 86-108, 114-135, 157-172, 177-183, 187-194,208-213, 218-226, 110-125 and 156-170 of Seq ID No 334; 5-24, 63-69,77-85, 94-112, 120-137, 140-146, 152-159, 166-172, 179-187, 193-199,206-212, 222-228, 234-240, 244-252, 257-264, 270-289, 298-309, 316-328,337-348, 363-375, 1-56 and 340-352 of Seq ID No 335; 18-39, 42-71,78-120, 124-144, 152-173, 179-189, 199-209, 213-222, 228-258, 269-304,329-361, 364-372, 374-389, 396-441 and 313-327 of Seq ID No 336; 19-25,91-98, 108-120, 156-162, 168-174, 191-204, 211-216, 232-266, 272-278,286-308, 316-321, 327-333, 344-355, 358-364, 384-391, 395-428, 464-476,487-495, 497-511, 544-561, 563-573, 575-582, 588-594, 10-25 and 322-338of Seq ID No 337; 14-26, 32-49, 51-57, 59-72, 80-91, 102-112, 119-125,147-161, 164-173, 175-183, 188-213, 217-222, 246-254, 260-276, 282-303,308-318, 321-328, 333-350, 352-359, 371-378, 392-401, 407-414, 416-443,448-463, 471-484, 490-497, 501-514, 519-527, 539-551, 557-570, 578-590,592-598, 600-610, 618-629, 633-647, 654-667, 676-689, 702-709, 718-726,728-737, 741-760, 764-780, 786-795, 808-826, 836-842, 845-852, 865-874,881-887, 931-945, 949-957, 968-974, 979-986, 1003-1009, 1023-1029 and90-103 of Seq ID No 338; 11-16, 37-56, 60-66, 69-77, 80-88, 93-106,117-139, 166-171 and 72-90 of Seq ID No 339; 59-84, 123-133, 145-150,161-167, 178-189 and 115-128 of Seq ID No 340; 15-33, 39-46, 52-64,74-87, 108-124, 127-144, 150-156, 173-179, 184-194, 201-208, 219-236,243-269, 272-295, 302-309, 343-349, 356-361, 370-379, 405-411, 414-423,430-451, 457-464, 466-475, 477-483, 496-502, 507-522, 541-548, 557-563,571-577, 579-585, 590-605, 626-642, 650-662, 671-691, 704-710, 751-769,775-781, 786-791, 794-829, 851-858, 868-878, 884-904, 913-919, 931-939and 132-142 of Seq ID No 341; 33-58, 64-71, 74-80, 83-88, 96-120,122-139, 146-157, 167-177, 207-213, 220-225, 236-242, 264-279, 300-305,326-336, 340-347, 350-360, 97-115 and 199-211 of Seq ID No 342; 4-26,43-57, 70-99, 102-117, 121-133, 142-148, 151-168, 170-183, 192-220,235-249, 258-279 and 30-41 of Seq ID No 343; 34-42, 48-58, 70-94,110-130, 154-160, 164-172, 178-183, 195-203, 211-222, 229-250, 256-261,274-284, 286-292, 312-323 and 222-233 of Seq ID No 344; 4-9, 15-36,38-45, 49-74, 78-88, 100-112, 136-191, 211-220, 226-233, 239-246,254-274, 287-307, 316-322, 342-353, 356-366, 373-378, 384-393, 405-431,449-457, 459-468, 487-511, 515-524, 529-541, 544-552, 562-568, 571-576and 208-280 of Seq ID No 345; 10-27, 31-37, 39-54, 71-108, 124-143 and2-107 of Seq ID No 346; 16-27, 38-57, 64-70, 90-102, 104-113, 116-137,160-166 and 1-80 of Seq ID No 347; 13-21, 31-36, 56-67, 127-136,153-171, 173-180, 184-200, 214-222, 225-231, 239-263, 267-273 and135-159 of Seq ID No 348; 12-27, 31-51, 68-74, 77-87, 94-101, 108-114,117-123, 127-134, 138-168, 173-196, 201-207, 212-217, 227-237, 247-257,264-280 and 205-223 of Seq ID No 349; 17-22, 25-54, 70-76, 92-100 and98-110 of Seq ID No 350; 7-29, 40-50, 60-67, 87-96, 105-111, 119-164,172-199, 206-212, 220-227, 237-259, 272-279, 282-293, 295-309, 313-319,321-328, 345-363, 376-386 and 159-176 of Seq ID No 351; 4-19, 24-30,36-43, 50-68, 71-89, 93-106, 141-152, 154-172, 179-197, 199-215,229-239, 246-252, 255-263, 281-298, 319-325, 329-356, 358-368, 374-390,397-409, 420-429, 432-444, 450-456, 459-475, 483-494, 496-502, 520-528,532-556 and 362-377 of Seq ID No 352; 18-25, 40-62, 77-85, 91-97,105-116, 123-133, 139-184, 189-197 and 122-140 of Seq ID No 353; 4-49,52-58, 62-70, 79-105, 109-133, 142-150, 163-168, 206-214, 220-228,233-240, 243-254, 274-281, 303-311, 327-338, 357-373, 378-396, 403-413,420-436, 441-453, 461-467, 475-481, 484-498, 506-512, 514-521, 523-529,562-579, 589-595, 598-603, 615-648, 714-722, 728-742, 749-758, 777-792,795-807 and 643-658 of Seq ID No 354; 8-27, 37-48, 51-56, 72-79, 87-106,120-138, 140-147, 167-176, 187-197, 205-216, 222-229, 234-239, 243-249,277-288, 292-315, 334-343, 347-353, 363-391, 398-404, 430-447, 461-467,478-492, 498-507 and 456-470 of Seq ID No 355; 5-12, 18-24, 59-69,80-93, 95-109, 119-125, 130-137, 139-147, 158-163, 168-176, 182-202,206-215, 222-239, 241-249, 267-277, 291-298, 311-318, 321-327, 338-344,348-355, 373-386, 393-406, 411-417, 434-443, 446-465, 473-484, 514-521,532-553, 584-594 and 221-237 of Seq ID No 356; 4-14, 27-34, 50-58,63-72, 79-106, 109-114, 121-142, 146-154, 161-167, 169-175, 178-201,223-238, 249-254, 259-264, 278-292, 294-312, 319-330 and 167-191 of SeqID No 357; 7-28, 36-42, 50-61, 63-80, 122-152, 161-174, 176-191 and140-190 of Seq ID No 358; 20-57, 59-65, 70-78, 86-102, 119-133, 142-161,163-173, 177-188, 192-202, 204-220, 222-236, 240-253, 279-319, 326-331,337-383, 390-399, 406-412, 420-427, 431-438 and 381-395 of Seq ID No359; 13-18, 28-34, 37-43, 50-59, 75-81, 83-97, 105-121, 139-147,200-206, 209-227, 231-247, 260-271, 318-327, 366-381, 388-394, 399-406and 182-201 of Seq ID No 360; 6-29, 37-43, 51-56, 70-77, 82-102,110-119, 127-143, 178-190, 201-209, 216-243, 261-269, 281-292, 305-313,327-339, 341-354, 356-373, 391-397, 423-429, 438-445, 450-478 and 21-314of Seq ID No 361; 4-12, 15-21, 32-41, 59-76, 80-89, 96-104 and 90-103 ofSeq ID No 362; 9-28, 30-41, 44-54, 69-74, 77-82, 90-97, 104-123,125-135, 149-155, 164-173, 177-184, 217-226, 230-235, 238-244, 258-272,282-297, 300-305, 309-315, 317-322, 327-336, 348-362, 368-374, 380-387,400-411, 414-424, 451-458, 460-466, 483-494, 497-503, 506-511, 521-528,540-553, 569-587, 598-606, 628-642, 661-681, 688-700, 718-733, 740-749,752-764, 769-783, 823-834, 848-854, 862-872, 878-884, 886-898, 915-920,938-951, 954-961, 963-972, 982-989, 996-1003, 1010-1016, 1021-1032,1038-1044, 1047-1057, 1060-1070, 1079-1088, 1094-1102, 1117-1127,1129-1135, 1142-1153, 1158-1204, 1212-1229, 1234-1263, 1269-1277,1308-1313, 1327-1338, 1344-1376, 1400-1415, 1436-1443, 1448-1458,1497-1504, 1511-1522, 1544-1566, 3-82 and 509-576 of Seq ID No 363;8-36, 40-64, 71-79, 88-94, 102-109, 118-127, 138-148, 151-159, 163-174,192-198, 200-206, 220-233, 268-273, 290-301, 304-309, 316-323, 331-349,378-391, 414-420, 427-437, 455-475, 494-510, 541-547, 549-555, 616-640,1-60, 55-139, 212-308, 386-458 and 458-624 of Seq ID No 364; 16-31,35-42, 70-77, 91-101, 120-130, 132-140, 143-153, 185-190, 195-202,215-222, 228-238, 241-251, 257-264, 268-277, 288-302, 312-324, 326-333,341-348, 364-382, 415-429, 438-454, 458-466, 491-499, 501-521 and273-281 of Seq ID No 365; 8-14, 32-57, 74-149, 155-177, 179-212,221-266, 271-296, 304-324, 329-346, 349-359, 368-401, 413-419, 426-454,465-478, 493-510 and 466-490 of Seq ID No 366; 22-28, 33-51, 64-89,96-119, 126-132, 138-146, 152-159, 161-169, 172-179, 193-198, 205-211,221-231, 235-254, 273-280, 297-303, 312-320, 328-346, 351-373, 378-384,391-398, 448-454, 460-468, 470-481, 516-558, 574-593, 597-602, 613-623,626-646, 649-656, 668-673, 675-683, 696-708, 715-722, 724-739, 745-751,759-777, 780-804, 816-822 and 102-113 of Seq ID No 367; 12-28, 41-91,98-107, 112-120, 125-131, 151-193, 215-221, 240-250, 263-280 and 128-138of Seq ID No 368; 16-24, 32-38, 46-62, 68-81, 90-105, 127-133, 144-150,160-166, 178-184, 186-202, 210-219, 232-240, 252-258, 264-273, 293-324,337-344, 349-357, 360-369, 385-398, 410-416, 419-427, 441-449, 458-476,508-515, 523-539, 544-549, 562-569, 571-579, 96-109 and 127-139 of SeqID No 369; 19-25, 28-34, 56-61, 85-97, 110-116 and 39-53 of Seq ID No370; 4-37, 41-50, 62-72, 91-97, 99-109, 114-125, 136-141, 149-158,160-166, 201-215 and 27-225 of Seq ID No 371; 15-31, 44-51, 96-105,122-130, 149-157, 162-168, 178-183, 185-192, 198-204, 206-213, 221-234,239-245, 248-255, 257-266, 289-335, 349-357, 415-422, 425-441, 448-454,462-468 and 463-481 of Seq ID No 372; 5-31, 39-55, 63-72, 76-99,106-155, 160-177, 179-199, 207-217, 223-240, 245-255, 261-267, 294-316,321-343, 354-378, 382-452, 477-488, 529-536, 555-569, 584-591, 593-612,620-627, 632-640, 647-654, 671-680, 698-704, 723-730, 732-750, 769-775,781-788, 822-852 and 505-525 of Seq ID No 373; 3-18 of Seq ID No 374;4-14 and 12-24 of Seq ID No 375; 4-11, 22-30 and 12-25 of Seq ID No 376;5-12 and 4-18 of Seq ID No 377; 4-28 and 7-14 of Seq ID No 378; 6-16 and8-16 of Seq ID No 379; 4-15, 18-33 and 24-36 of Seq ID No 380; 4-10,16-21 and 20-31 of Seq ID No 381; 6-19 of Seq ID No 382; 11-18 and 3-10of Seq ID No 383; 13-24 and 3-15 of Seq ID No 384; 15-27 and 7-16 of SeqID No 385; 11-16 and 1-15 of Seq ID No 386; 4-16 and 9-21 of Seq ID No387; 4-24, 40-48, 54-67 and 22-39 of Seq ID No 388; 6-30, 34-55, 62-68,78-106 and 68-74 of Seq ID No 389; 3-14 of Seq ID No 390; 9-19 and 6-21of Seq ID No 391; 4-17 and 1-9 of Seq ID No 392; 5-30 and 1-8 of Seq IDNo 393; 4-16, 23-46, 51-56 and 45-55 of Seq ID No 394; 7-16 of Seq ID No395; 2-14 of Seq ID No 396; 4-36, 43-65 and 50-62 of Seq ID No 397;10-30 and 14-21 of Seq ID No 398; 9-17 and 1-10 of Seq ID No 399; 4-12and 3-16 of Seq ID No 400; 4-15 and 5-23 of Seq ID No 401; 10-21 of SeqID No 402; 6-16 of Seq ID No 403; 4-29, 31-38 and 2-14 of Seq ID No 404;4-35 and 33-42 of Seq ID No 405; 2-17 of Seq ID No 406; 9-18, 30-35 and15-33 of Seq ID No 407; 4-9 and 6-12 of Seq ID No 408; 3-17 of Seq ID No409; 12-21, 37-44, 52-61, 72-80 and 38-48 of Seq ID No 410; 4-10, 29-44,54-61, 69-78 and 13-27 of Seq ID No 411; 13-23, 36-53 and 2-15 of Seq IDNo 412; 4-25, 28-46, 56-72, 81-99, 120-132, 134-142, 154-160 and 129-141of Seq ID No 413; 4-15, 24-33, 35-41, 64-86 and 21-33 of Seq ID No 414;9-15 and 4-13 of Seq ID No 415; 4-11, 13-19, 34-48 and 15-32 of Seq IDNo 416; 4-21 and 11-31 of Seq ID No 417; 23-57 and 38-50 of Seq ID No418; 4-32 and 3-13 of Seq ID No 419; 4-10, 13-25, 32-42, 56-68, 72-84and 26-38 of Seq ID No 420; 4-20, 31-48, 52-58, 65-71, 80-93, 99-108,114-123 and 37-49 of Seq ID No 421; 6-12, 14-20 and 3-25 of Seq ID No422; 14-25, 27-38 and 5-14 of Seq ID No 423; 4-41, 57-105, 109-118,123-136, 144-152 and 86-99 of Seq ID No 424; 6-19 of Seq ID No 425; 2-19of Seq ID No 426; 14-47 and 1-14 of Seq ID No 427; 4-21, 29-44 and 2-18of Seq ID No 428; 23-29 and 10-28 of Seq ID No 429; 6-16, 22-36 and11-22 of Seq ID No 430; 4-19, 30-44 and 18-27 of Seq ID No 431; 5-15,37-45, 58-65 and 38-47 of Seq ID No 432; 4-15, 23-34 and 4-15 of Seq IDNo 433; 30-36, 44-54, 79-85, 101-114, 138-152, 154-164, 170-175,179-200, 213-220, 223-240, 243-255, 258-264, 268-284 and 10-28 of Seq IDNo 434; the peptides comprising amino acid sequences of column“Identical region” of the Table 1B, especially peptides comprising aminoacid 210-226 and 738-753 of Seq ID No 449; 326-344, 326-348, 338-354,371-392, 801-809 and 877-901 of Seq ID No 450; 893-906 of Seq ID No 451;51-69 of Seq ID No 452; 110-125 of Seq ID No 453; 291-305 of Seq ID No454; 210-226 and 738-753 of Seq ID No 455; 326-344, 326-348, 338-354,371-392, 801-809 and 877-901 of Seq ID No 456; 893-906 of Seq ID No 457;51-69 of Seq ID No 458; 110-125 of Seq ID No 459; 291-305 of Seq ID No460; 32-44 of Seq ID No 461; 399-410 of Seq ID No 462; the serumreactive epitopes as specified in the column of “aa from” to “aa to” ofTable 2, especially peptides comprising amino acid 120-143, 138-161 and156-179 of Seq ID No 218; 110-129 and 168-184 of Seq ID No 219; 74-90 ofSeq ID No 222; 759-773 of Seq ID No 223; 237-260 of Seq ID No 224;265-284 of Seq ID No 225; 65-74 of Seq ID No 226; 41-50 of Seq ID No227; 163-174 of Seq ID No 229; 26-37 of Seq ID No 230; 174-189 of Seq IDNo 232; 240-256 of Seq ID No 234; 285-297 of Seq ID No 236; 238-247 ofSeq ID No 238; 491-519 of Seq ID No 239; 114-140 of Seq ID No 243;267-284 of Seq ID No 250; 439-453 of Seq ID No 252; 162-178 of Seq ID No253; 347-364 of Seq ID No 254; 699-715 of Seq ID No 255; 60-71 of Seq IDNo 256; 244-257 of Seq ID No 257; 44-63 and 57-76 of Seq ID No 258;185-196 of Seq ID No 260; 119-129 of Seq ID No 263; 182-195 of Seq ID No266; 32-44 and 424-442 of Seq ID No 267; 247-256 of Seq ID No 268;678-694, 785-805, 55-77 and 72-94 of Seq ID No 269; 210-226 of Seq ID No281; 37-59 of Seq ID No 289; 13-29 of Seq ID No 296; 136-159 of Seq IDNo 348; 205-222 of Seq ID No 349; 99-110 of Seq ID No 350; 160-176 ofSeq ID No 351; 457-470 of Seq ID No 355; 221-237 of Seq ID No 356;167-190 of Seq ID No 357; 96-120 of Seq ID No 361; 399-417, 503-519 and544-563 of Seq ID No 364; 46-68, 159-183 and 184-198 of Seq ID No 371;463-481 of Seq ID No 372; the immunogenic epitopes as specified in thecolumn of “aa from” to “aa to” of Table 4; especially peptidescomprising amino acid 110-129 and 168-184 of Seq ID No 219; 877-901,333-354, 326-344 and 801-809 of Seq ID No 277; 1-54 of Seq ID No 347;544-563, 31-51, 107-119, 399-417 and 503-519 of Seq ID No 364; 120-198of Seq ID No 218; 20-35 of Seq ID No 219; 118-201 of Seq ID No 221;48-132 of Seq ID No 242; 118-136 of Seq ID No 249; 162-178 of Seq ID No253; 347-364 of Seq ID No 254; 699-715 of Seq ID No 255; 50-76 of Seq IDNo 258; 785-819 and 44-128 of Seq ID No 269; 90-128 of Seq ID No 274;314-384 of Seq ID No 289; 327-349 of Seq ID No 293; 242-314, 405-478 and23-100 of Seq ID No 304; 129-210 of Seq ID No 305; 162-188 of Seq ID No307; 750-772 of Seq ID No 310; 1-56 of Seq ID No 335; 322-337 of Seq IDNo 337; 72-90 of Seq ID No 339; 374-395 of Seq ID No 345; 136-159 of SeqID No 348; 141-164 of Seq ID No 358; 96-157 of Seq ID No 361; 1-82 ofSeq ID No 363; 489-556 of Seq ID No 364; 159-183 and 49-133 of Seq ID No371; The peptides comprising amino acid sequences of column “predictedimmunogenic aa” and “location of identified immunogenic region (aa)” ofTable 5, especially peptides comprising amino acid 4-26, 35-41, 53-61,73-84, 103-108, 114-120, 140-146, 156-162, 192-208, 214-219, 227-233,239-252, 260-268, 284-297, 1-48 and 113-133 of Seq ID No 475; 4-27,38-44, 50-56, 59-64, 72-79, 83-89, 92-97, 108-116, 123-148, 152-167,183-196, 200-220, 232-244, 255-261, 265-274, 282-302, 309-317, 1-79 and231-302 of Seq ID No 476; 6-28, 66-72, 85-105, 115-121, 144-151,160-170, 176-185, 223-230, 252-288, 296-310, 319-333, 367-374, 458-464,471-480, 483-488, 520-528, 530-549, 559-564, 593-601, 606-616, 636-643,655-662, 676-682, 684-699, 719-726, 735-750, 757-764, 777-785, 799-810,812-843, 846-853, 868-873, 880-889, 891-899, 909-929, 934-940, 963-969,998-1004, 1007-1014, 1016-1022, 1030-1046, 1-80 and 808-821 of Seq ID No477; 7-24, 35-41, 75-81, 91-114, 122-132, 137-144, 148-156, 183-192,194-200, 212-228, 233-238, 251-258, 275-295, 326-332, 337-346, 1-79 and305-321 of Seq ID No 478; 31-38, 42-52, 66-72, 86-92, 98-104, 115-122,127-146, 154-164, 169-187, 198-212, 225-237, 255-269, 13-92 and 135-142of Seq ID No 479; 4-36, 39-49, 63-69, 71-77, 81-88, 123-131, 133-139,160-169, 174-180, 188-194, 210-217, 273-278, 289-300, 317-334, 336-341,383-401, 425-438, 1-68, 212-270 and 402-446 of Seq ID No 480; 21-29,31-42, 49-63, 72-79, 81-93, 112-132, 159-165, 188-195, 197-232, 262-267,279-286, 294-301, 318-326, 348-366, 381-405, 409-426, 436-465, 471-480,484-492, 497-505, 521-544, 554-561, 567-577, 581-589, 601-609, 611-622,636-651, 653-667, 669-685, 700-708, 716-722, 729-744, 749-766, 780-786,789-811, 814-864, 1-57 and 84-106 of Seq ID No 481; 6-24, 35-48, 57-63,72-78, 87-92, 113-119, 123-137, 147-153, 173-181, 212-233 and 1-124 ofSeq ID No 482; 13-34, 62-69, 78-83, 86-91, 98-104, 107-115, 146-159,179-188, 195-205, 209-221, 226-233, 239-253, 276-282, 284-294, 297-308,331-354, 375-382, 388-399, 421-433, 449-458, 464-469, 472-491, 508-513,525-531, 534-550, 575-593, 601-618, 629-635, 654-661, 666-680, 706-721,723-740, 771-805, 810-830, 845-851 and 1-84 of Seq ID No 483; 4-32,45-64, 73-83, 86-92, 100-111, 125-147, 157-163, 170-175, 177-188,226-232, 245-252, 258-274, 320-335, 348-359 and 1-71 of Seq ID No 484;13-40, 43-71, 76-83, 87-101, 109-119, 125-156, 162-175, 182-219,226-232, 240-262, 270-287, 306-318, 326-342, 344-408, 414-444, 449-456and 1-51 of Seq ID No 485; 4-16, 18-34, 45-54, 99-108, 134-140, 203-212,241-257, 266-274, 279-291, 308-315, 330-336, 355-370, 374-382, 402-410,428-455, 466-472, 474-480, 531-554, 560-566, 572-580, 597-618, 632-660,664-674, 676-685, 691-705, 708-735, 750-768, 1-87 and 342-480 of Seq IDNo 486; The serum reactive epitopes as specified in the column of “aafrom” to “aa to” of Table 6, especially peptides comprising amino acid115-132 and 1-26 of Seq ID No 475; 33-55 of Seq ID No 476; 1-25 of SeqID No 478; 37-61 of Seq ID No 479; 1-24 of Seq ID No 480; 1-23 of Seq IDNo 481; 46-60 of Seq ID No 482; 1-28, 23-50 and 45-71 of Seq ID No 483;1-22 and 17-38 of Seq ID No 484; 1-22 and 17-38 of Seq ID No 485; 1-27,22-47 and 422-447 of Seq ID No 486; The immunogenic epitopes asspecified in the column of “aa from” to “aa to” of Table 7, especiallypeptides comprising amino acid 115-132 and 1-47 of Seq ID No 475; 1-55of Seq ID No 476; 22-85 of Seq ID No 477; 307-320 and 1-44 of Seq ID No478; 15-76 and 40-92 of Seq ID No 479; 1-59, 213-269 and 403-445 of SeqID No 480; 1-56 and 85-105 of Seq ID No 481; 37-121 of Seq ID No 482;1-71 of Seq ID No 483; 1-38 of Seq ID No 484; 1-38 of Seq ID No 485;1-47 of Seq ID No 486.

The present invention also provides a process for producing a S.agalactiae hyperimmune serum reactive antigen or a fragment thereofaccording to the present invention comprising expressing one or more ofthe nucleic acid molecules according to the present invention in asuitable expression system.

Moreover, the present invention provides a process for producing a cell,which expresses a S agalactiae hyperimmune serum reactive antigen or afragment thereof according to the present invention comprisingtransforming or transfecting a suitable host cell with the vectoraccording to the present invention.

According to the present invention a pharmaceutical composition,especially a vaccine, comprising a hyperimmune serum-reactive antigen ora fragment thereof as defined in the present invention or a nucleic acidmolecule as defined in the present invention is provided.

In a preferred embodiment the pharmaceutical composition furthercomprises an immunostimulatory substance, preferably selected from thegroup comprising polycationic polymers, especially polycationicpeptides, immunostimulatory deoxynucleotides (ODNs), peptides containingat least two LysLeuLys motifs, especially KLKL5KLK (SEQ ID NO:487),neuroactive compounds, especially human growth hormone, alumn, Freund'scomplete or incomplete adjuvants or combinations thereof.

In a more preferred embodiment the immunostimulatory substance is acombination of either a polycationic polymer and immunostimulatorydeoxynucleotides or of a peptide containing at least two LysLeuLysmotifs and immunostimulatory deoxynucleotides.

In a still more preferred embodiment the polycationic polymer is apolycationic peptide, especially polyarginine.

According to the present invention the use of a nucleic acid moleculeaccording to the present invention or a hyperimmune serum-reactiveantigen or fragment thereof according to the present invention for themanufacture of a pharmaceutical preparation, especially for themanufacture of a vaccine against S. agalactiae infection, is provided.

Also an antibody, or at least an effective part thereof, which binds atleast to a selective part of the hyperimmune serum-reactive antigen or afragment thereof according to the present invention, is providedherewith.

In a preferred embodiment the antibody is a monoclonal antibody.

In another preferred embodiment the effective part of the antibodycomprises Fab fragments.

In a further preferred embodiment the antibody is a chimeric antibody.

In a still preferred embodiment the antibody is a humanized antibody.

The present invention also provides a hybridoma cell line, whichproduces an antibody according to the present invention.

Moreover, the present invention provides a method for producing anantibody according to the present invention, characterized by thefollowing steps:

-   -   initiating an immune response in a non-human animal by        administrating an hyperimmune serum-reactive antigen or a        fragment thereof, as defined in the invention, to said animal,    -   removing an antibody containing body fluid from said animal, and    -   producing the antibody by subjecting said antibody containing        body fluid to further purification steps.

Accordingly, the present invention also provides a method for producingan antibody according to the present invention, characterized by thefollowing steps:

-   -   initiating an immune response in a non-human animal by        administrating an hyperimmune serum-reactive antigen or a        fragment thereof, as defined in the present invention, to said        animal,    -   removing the spleen or spleen cells from said animal,    -   producing hybridoma cells of said spleen or spleen cells,    -   selecting and cloning hybridoma cells specific for said        hyperimmune serum-reactive antigens or a fragment thereof,    -   producing the antibody by cultivation of said cloned hybridoma        cells and optionally further purification steps.

The antibodies provided or produced according to the above methods maybe used for the preparation of a medicament for treating or preventingS. agalactiae infections.

According to another aspect the present invention provides anantagonist, which binds to a hyperimmune serum-reactive antigen or afragment thereof according to the present invention.

Such an antagonist capable of binding to a hyperimmune serum-reactiveantigen or fragment thereof according to the present invention may beidentified by a method comprising the following steps:

-   -   a) contacting an isolated or immobilized hyperimmune        serum-reactive antigen or a fragment thereof according to the        present invention with a candidate antagonist under conditions        to permit binding of said candidate antagonist to said        hyperimmune serum-reactive antigen or fragment, in the presence        of a component capable of providing a detectable signal in        response to the binding of the candidate antagonist to said        hyperimmune serum reactive antigen or fragment thereof; and    -   b) detecting the presence or absence of a signal generated in        response to the binding of the antagonist to the hyperimmune        serum reactive antigen or the fragment thereof.

An antagonist capable of reducing or inhibiting the interaction activityof a hyperimmune serum-reactive antigen or a fragment thereof accordingto the present invention to its interaction partner may be identified bya method comprising the following steps:

-   -   a) providing a hyperimmune serum reactive antigen or a        hyperimmune fragment thereof according to the present invention,    -   b) providing an interaction partner to said hyperimmune serum        reactive antigen or a fragment thereof, especially an antibody        according to the present invention,    -   c) allowing interaction of said hyperimmune serum reactive        antigen or fragment thereof to said interaction partner to form        an interaction complex,    -   d) providing a candidate antagonist,    -   e) allowing a competition reaction to occur between the        candidate antagonist and the interaction complex,    -   f) determining whether the candidate antagonist inhibits or        reduces the interaction activities of the hyperimmune serum        reactive antigen or the fragment thereof with the interaction        partner.

The hyperimmune serum reactive antigens or fragments thereof accordingto the present invention may be used for the isolation and/orpurification and/or identification of an interaction partner of saidhyperimmune serum reactive antigen or fragment thereof.

The present invention also provides a process for in vitro diagnosing adisease related to expression of a hyperimmune serum-reactive antigen ora fragment thereof according to the present invention comprisingdetermining the presence of a nucleic acid sequence encoding saidhyperimmune serum reactive antigen or fragment thereof according to thepresent invention or the presence of the hyperimmune serum reactiveantigen or fragment thereof according to the present invention.

The present invention also provides a process for in vitro diagnosis ofa bacterial infection, especially a S. agalactiae infection, comprisinganalyzing for the presence of a nucleic acid sequence encoding saidhyperimmune serum reactive antigen or fragment thereof according to thepresent invention or the presence of the hyperimmune serum reactiveantigen or fragment thereof according to the present invention.

Moreover, the present invention provides the use of a hyperimmune serumreactive antigen or fragment thereof according to the present inventionfor the generation of a peptide binding to said hyperimmune serumreactive antigen or fragment thereof, wherein the peptide is ananticaline.

The present invention also provides the use of a hyperimmuneserum-reactive antigen or fragment thereof according to the presentinvention for the manufacture of a functional nucleic acid, wherein thefunctional nucleic acid is selected from the group comprising aptamersand spiegelmers.

The nucleic acid molecule according to the present invention may also beused for the manufacture of a functional ribonucleic acid, wherein thefunctional ribonucleic acid is selected from the group comprisingribozymes, antisense nucleic acids and siRNA.

The present invention advantageously provides an efficient, relevant andcomprehensive set of isolated nucleic acid molecules and their encodedhyperimmune serum reactive antigens or fragments thereof identified fromS. agalactiae using an antibody preparation from multiple human plasmapools and surface expression libraries derived from the genome of S.agalactiae. Thus, the present invention fulfils a widely felt demand forS. agalactiae antigens, vaccines, diagnostics and products useful inprocedures for preparing antibodies and for identifying compoundseffective against S. agalactiae infection.

An effective vaccine should be composed of proteins or polypeptides,which are expressed by all strains and are able to induce high affinity,abundant antibodies against cell surface components of S. agalactiae.The antibodies should be IgG1 and/or IgG3 for opsonization, and any IgGsubtype and IgA for neutralisation of adherence and toxin action. Achemically defined vaccine must be definitely superior compared to awhole cell vaccine (attenuated or killed), since components of S.agalactiae, which cross-react with human tissues or inhibit opsonizationcan be eliminated, and the individual proteins inducing protectiveantibodies and/or a protective immune response can be selected.

The approach, which has been employed for the present invention, isbased on the interaction of GBS proteins or peptides with the antibodiespresent in human sera. The antibodies produced against S. agalactiae bythe human immune system and present in human sera are indicative of thein vivo expression of the antigenic proteins and their immunogenicity.In addition, the antigenic proteins as identified by the bacterialsurface display expression libraries using pools of pre-selected sera,are processed in a second and third round of screening by individualselected or generated sera. Thus the present invention supplies anefficient, relevant, comprehensive set of GBS antigens as apharmaceutical composition, especially a vaccine preventing infection byS. agalactiae.

In the antigen identification program for identifying a comprehensiveset of antigens according to the present invention, at least twodifferent bacterial surface expression libraries are screened withseveral serum pools or plasma fractions or other pooled antibodycontaining body fluids (antibody pools). The antibody pools are derivedfrom a serum collection, which has been tested against antigeniccompounds of S. agalactiae, such as whole cell extracts and culturesupernatant proteins. Preferably, three distinct serum collections areused, obtained ad 1. from healthy pregnant women tested negative forcervical and anorectal carriage of GBS, ad 2. healthy pregnant womentested positive for cervical and/or anorectal carriage of GBS who'snewborn remained GBS-free (although with antibiotic prevention), ad 3.from adults below <45 years of age without clinical disease. Sera haveto react with multiple GBS-specific antigens in order to be consideredhyperimmune and therefore relevant in the screening method applied forthe present invention.

The expression libraries as used in the present invention should allowexpression of all potential antigens, e.g. derived from all secreted andsurface proteins of S. agalactiae. Bacterial surface display librarieswill be represented by a recombinant library of a bacterial hostdisplaying a (total) set of expressed peptide sequences of S. agalactiaeon two selected outer membrane proteins (LamB and FhuA) at the bacterialhost membrane {Georgiou, G., 1997}; {Etz, H. et al., 2001}. One of theadvantages of using recombinant expression libraries is that theidentified hyperimmune serum-reactive antigens may be instantly producedby expression of the coding sequences of the screened and selectedclones expressing the hyperimmune serum-reactive antigens withoutfurther recombinant DNA technology or cloning steps necessary.

The comprehensive set of antigens identified by the described programaccording to the present invention is analyzed further by one or moreadditional rounds of screening. Therefore individual antibodypreparations or antibodies generated against selected peptides, whichwere identified as immunogenic are used. According to a preferredembodiment the individual antibody preparations for the second round ofscreening are derived from pregnant women and non-pregant adults whoshow an antibody titer above a certain minimum level, for example anantibody titer being higher than 80 percentile, preferably higher than90 percentile, especially higher than 95 percentile of the human(patient or healthy individual) sera tested. Using such high titerindividual antibody preparations in the second screening round allows avery selective identification of the hyperimmune serum-reactive antigensand fragments thereof from S. agalactiae.

Following the comprehensive screening procedure, the selected antigenicproteins, expressed as recombinant proteins or in vitro translatedproducts, in case it can not be expressed in prokaryotic expressionsystems, or the identified antigenic peptides (produced synthetically)are tested in a second screening by a series of ELISA and Westernblotting assays for the assessment of their immunogenicity with a largehuman serum collection (minimum ˜150 healthy and patients sera).

It is important that the individual antibody preparations (which mayalso be the selected serum) allow a selective identification of the mostpromising candidates of all the hyperimmune serum-reactive antigens fromall the promising candidates from the first round. Therefore, preferablyat least 10 individual antibody preparations (i.e. antibody preparations(e.g. sera) from at least 10 different individuals having suffered froman infection to the chosen pathogen) should be used in identifying theseantigens in the second screening round. Of course, it is possible to usealso less than 10 individual preparations, however, selectivity of thestep may not be optimal with a low number of individual antibodypreparations. On the other hand, if a given hyperimmune serum-reactiveantigen (or an antigenic fragment thereof) is recognized by at least 10individual antibody preparations, preferably at least 30, especially atleast 50 individual antibody preparations, identification of thehyperimmune serum-reactive antigen is also selective enough for a properidentification. Hyperimmune serum-reactivity may of course be testedwith as many individual preparations as possible (e.g. with more than100 or even with more than 1,000).

Therefore, the relevant portion of the hyperimmune serum-reactiveantibody preparations according to the method of the present inventionshould preferably be at least 10, more preferred at least 30, especiallyat least 50 individual antibody preparations. Alternatively (or incombination) hyperimmune serum-reactive antigens may preferably be alsoidentified with at least 20%, preferably at least 30%, especially atleast 40% of all individual antibody preparations used in the secondscreening round.

According to a preferred embodiment of the present invention, the serafrom which the individual antibody preparations for the second round ofscreening are prepared (or which are used as antibody preparations), areselected by their titer against S. agalactiae (e.g. against apreparation of this pathogen, such as a lysate, cell wall components andrecombinant proteins). Preferably, some are selected with a total IgAtiter above 300 U, especially above 500 U, and/or an IgG titer above5,000 U, especially above 10,000 U (U=units, calculated from theOD_(405 nm) reading at a given dilution) when the whole organism (totallysate or whole cells) is used as antigen in the ELISA.

The antibodies produced against streptococci by the human immune systemand present in human sera are indicative of the in vivo expression ofthe antigenic proteins and their immunogenicity. The recognition oflinear epitopes recognized by serum antibodies can be based on sequencesas short as 4-5 amino acids. Of course it does not necessarily mean thatthese short peptides are capable of inducing the given antibody in vivo.For that reason the defined epitopes, polypeptides and proteins arefurther to be tested in animals (mainly in mice) for their capacity toinduce antibodies against the selected proteins in vivo.

The preferred antigens are located on the cell surface or secreted, andare therefore accessible extracellularly. Antibodies against cell wallproteins are expected to serve multiple purposes: to inhibit adhesion,to interfere with nutrient acquisition, to inhibit immune evasion nandto promote phagocytosis {Hornef, M. et al., 2002}. Antibodies againstsecreted proteins are beneficial in neutralisation of their function astoxin or virulence component. It is also known that bacteria communicatewith each other through secreted proteins. Neutralizing antibodiesagainst these proteins will interrupt growth-promoting cross-talkbetween or within streptococcal species. Bioinformatic analyzes (signalsequences, cell wall localisation signals, transmembrane domains) provedto be very useful in assessing cell surface localisation or secretion.The experimental approach includes the isolation of antibodies with thecorresponding epitopes and proteins from human serum, and the generationof immune sera in mice against (poly) peptides selected by the bacterialsurface display screens. These sera are then used in a third round ofscreening as reagents in the following assays: cell surface staining ofS. agalactiae grown under different conditions (FACS or microscopy),determination of neutralizing capacity (toxin, adherence), and promotionof opsonization and phagocytosis (in vitro phagocytosis assay).

For that purpose, bacterial E. coli clones are directly injected intomice and immune sera are taken and tested in the relevant in vitro assayfor functional opsonic or neutralizing antibodies. Alternatively,specific antibodies may be purified from human or mouse sera usingpeptides or proteins as substrate.

Host defence against S. agalactiae relies mainly on opsonophagocytickilling mechanism. Inducing high affinity antibodies of the opsonic andneutralizing type by vaccination helps the innate immune system toeliminate bacteria and toxins. This makes the method according to thepresent invention an optimal tool for the identification of GBSantigenic proteins.

The skin and mucous membranes are formidable barriers against invasionby streptococci. However, once the skin or the mucous membranes arebreached the first line of non-adaptive cellular defence begins itsco-ordinate action through complement and phagocytes, especially thepolymorphonuclear leukocytes (PMNs). These cells can be regarded as thecornerstones in eliminating invading bacteria. As Streptococcusagalactiae is a primarily extracellular pathogen, the majoranti-streptococcal adaptive response comes from the humoral arm of theimmune system, and is mediated through three major mechanisms: promotionof opsonization, toxin neutralisation, and inhibition of adherence. Itis believed that opsonization is especially important, because of itsrequirement for an effective phagocytosis. For efficient opsonizationthe microbial surface has to be coated with antibodies and complementfactors for recognition by PMNs through receptors to the Fc fragment ofthe IgG molecule or to activated C3b. After opsonization, streptococciare phagocytosed and killed. Antibodies bound to specific antigens onthe cell surface of bacteria serve as ligands for the attachment to PMNsand to promote phagocytosis. The very same antibodies bound to theadhesins and other cell surface proteins are expected to neutralizeadhesion and prevent colonization. The selection of antigens as providedby the present invention is thus well suited to identify those that willlead to protection against infection in an animal model or in humans.

According to the antigen identification method used herein, the presentinvention can surprisingly provide a set of comprehensive novel nucleicacids and novel hyperimmune serum reactive antigens and fragmentsthereof of S. agalactiae, among other things, as described below.According to one aspect, the invention particularly relates to thenucleotide sequences encoding hyperimmune serum reactive antigens whichsequences are set forth in the Sequence listing Seq ID No: 1-217,435-448 and 463-474 and the corresponding encoded amino acid sequencesrepresenting hyperimmune serum reactive antigens are set forth in theSequence Listing Seq ID No 218-434, 449-462 and 475-486.

In a preferred embodiment of the present invention, a nucleic acidmolecule is provided which exhibits 70% identity over their entirelength to a nucleotide sequence set forth with Seq ID No 14, 90,157-216. Most highly preferred are nucleic acids that comprise a regionthat is at least 80% or at least 85% identical over their entire lengthto a nucleic acid molecule set forth with Seq ID No 14, 90, 157-216. Inthis regard, nucleic acid molecules at least 90%, 91%, 92%, 93%, 94%,95%, or 96% identical over their entire length to the same areparticularly preferred. Furthermore, those with at least 97% are highlypreferred, those with at least 98% and at least 99% are particularlyhighly preferred, with at least 99% or 99.5% being the more preferred,with 100% identity being especially preferred. Moreover, preferredembodiments in this respect are nucleic acids which encode hyperimmuneserum reactive antigens or fragments thereof (polypeptides) which retainsubstantially the same biological function or activity as the maturepolypeptide encoded by said nucleic acids set forth in the Seq ID No 14,90, 157-216.

Identity, as known in the art and used herein, is the relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. Identity canbe readily calculated. While there exist a number of methods to measureidentity between two polynucleotide or two polypeptide sequences, theterm is well known to skilled artisans (e.g. Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity are codifiedin computer programs. Preferred computer program methods to determineidentity between two sequences include, but are not limited to, GCGprogram package {Devereux, J. et al., 1984}, BLASTP, BLASTN, and FASTA{Altschul, S. et al., 1990}.

According to another aspect of the invention, nucleic acid molecules areprovided which exhibit 96% or more than 96%, especially 100% sequenceidentity to the nucleic acid sequence set forth with Seq ID No 1, 3,5-13, 15, 18-25, 27-31, 33-36, 39-68, 70-85, 92-100, 103-126, 128-145,147, 149-156, 217, 435-448 and 463-474.

According to a further aspect of the present invention, nucleic acidmolecules having 98% or more than 98%, especially 100% sequence identityto a nucleic acid molecule are provided which are identical to thenucleic acid sequences set forth with Seq ID No 32, 86, 91, 101, 127.

The nucleic acid molecules according to the present invention can as asecond alternative also be a nucleic acid molecule which is at leastessentially complementary to the nucleic acid described as the firstalternative above. As used herein complementary means that a nucleicacid strand is base pairing via Watson-Crick base pairing with a secondnucleic acid strand. Essentially complementary as used herein means thatthe base pairing is not occurring for all of the bases of the respectivestrands but leaves a certain number or percentage of the bases unpairedor wrongly paired. The percentage of correctly pairing bases ispreferably at least 70%, more preferably 80%, even more preferably 90%and most preferably any percentage higher than 90%. It is to be notedthat a percentage of 70% matching bases is considered as homology andthe hybridization having this extent of matching base pairs isconsidered as stringent. Hybridization conditions for this kind ofstringent hybridization may be taken from Current Protocols in MolecularBiology (John Wiley and Sons, Inc., 1987). More particularly, thehybridization conditions can be as follows:

-   -   Hybridization performed e.g. in 5×SSPE, 5×Denhardt's reagent,        0.1% SDS, 100 g/mL sheared DNA at 68° C.    -   Moderate stringency wash in 0.2×SSC, 0.1% SDS at 42° C.    -   High stringency wash in 0.1×SSC, 0.1% SDS at 68° C.

Genomic DNA with a GC content of 50% has an approximate T_(M) of 96° C.For 1% mismatch, the T_(M) is reduced by approximately 1° C.

In addition, any of the further hybridization conditions describedherein are in principle applicable as well.

Of course, all nucleic acid sequence molecules which encode the samepolypeptide molecule as those identified by the present invention areencompassed by any disclosure of a given coding sequence, since thedegeneracy of the genetic code is directly applicable to unambiguouslydetermine all possible nucleic acid molecules which encode a givenpolypeptide molecule, even if the number of such degenerated nucleicacid molecules may be high. This is also applicable for fragments of agiven polypeptide, as long as the fragments encode a polypeptide beingsuitable to be used in a vaccination connection, e.g. as an active orpassive vaccine.

The nucleic acid molecule according to the present invention can as athird alternative also be a nucleic acid which comprises a stretch of atleast 15 bases of the nucleic acid molecule according to the first andsecond alternative of the nucleic acid molecules according to thepresent invention as outlined above. Preferably, the bases form acontiguous stretch of bases. However, it is also within the scope of thepresent invention that the stretch consists of two or more moieties,which are separated by a number of bases.

The present nucleic acids may preferably consist of at least 20, evenmore preferred at least 30, especially at least 50 contiguous bases fromthe sequences disclosed herein. The suitable length may easily beoptimized due to the planned area of use (e.g. as (PCR) primers, probes,capture molecules (e.g. on a (DNA) chip), etc.). Preferred nucleic acidmolecules contain at least a contiguous 15 base portion of one or moreof the predicted immunogenic amino acid sequences listed in tables 1 and2, especially the sequences of table 2 with scores of more than 10,preferably more than 20, especially with a score of more than 25.Specifically preferred are nucleic acids containing a contiguous portionof a DNA sequence of any sequence in the sequence protocol of thepresent application which shows 1 or more, preferably more than 2,especially more than 5, non-identical nucleic acid residues compared tothe published Streptococcus agalactiae strain NEM316 (ATCC 12403) genome({Glaser, P. et al., 2002}; GenBank accession AL732656) and/or any otherpublished S. agalactiae genome sequence or parts thereof, especially ofthe serotype V 2603 V/R (A909) strain {Tettelin, H. et al., 2002});GenBank accession AE009948). Specifically preferred non-identicalnucleic acid residues are residues, which lead to a non-identical aminoacid residue. Preferably, the nucleic acid sequences encode polypeptideshaving at least 1, preferably at least 2, preferably at least 3different amino acid residues compared to the published S. agalactiaecounterparts mentioned above. Also such isolated polypeptides, beingfragments of the proteins (or the whole protein) mentioned herein e.g.in the sequence listing, having at least 6, 7, or 8 amino acid residuesand being encoded by these nucleic acids are preferred.

The nucleic acid molecule according to the present invention can as afourth alternative also be a nucleic acid molecule which anneals understringent hybridisation conditions to any of the nucleic acids of thepresent invention according to the above outlined first, second, andthird alternative. Stringent hybridisation conditions are typicallythose described herein.

Finally, the nucleic acid molecule according to the present inventioncan as a fifth alternative also be a nucleic acid molecule which, butfor the degeneracy of the genetic code, would hybridize to any of thenucleic acid molecules according to any nucleic acid molecule of thepresent invention according to the first, second, third, and fourthalternative as outlined above. This kind of nucleic acid molecule refersto the fact that preferably the nucleic acids according to the presentinvention code for the hyperimmune serum reactive antigens or fragmentsthereof according to the present invention. This kind of nucleic acidmolecule is particularly useful in the detection of a nucleic acidmolecule according to the present invention and thus the diagnosis ofthe respective microorganisms such as S. agalactiae and any disease ordiseased condition where this kind of microorganims is involved.Preferably, the hybridisation would occur or be preformed understringent conditions as described in connection with the fourthalternative described above.

Nucleic acid molecule as used herein generally refers to any ribonucleicacid molecule or deoxyribonucleic acid molecule, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, nucleic acidmolecule as used herein refers to, among other, single-anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded RNA, and RNA that is a mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded, or a mixture of single- and double-stranded regions. Inaddition, nucleic acid molecule as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide. Asused herein, the term nucleic acid molecule includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“nucleic acid molecule” as that term is intended herein. Moreover, DNAsor RNAs comprising unusual bases, such as inosine, or modified bases,such as tritylated bases, to name just two examples, are nucleic acidmolecule as the term is used herein. It will be appreciated that a greatvariety of modifications have been made to DNA and RNA that serve manyuseful purposes known to those of skill in the art. The term nucleicacid molecule as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of nucleic acid molecule,as well as the chemical forms of DNA and RNA characteristic of virusesand cells, including simple and complex cells, inter alia. The termnucleic acid molecule also embraces short nucleic acid molecules oftenreferred to as oligonucleotide(s). “Polynucleotide” and “nucleic acid”or “nucleic acid molecule” are often used interchangeably herein.

Nucleic acid molecules provided in the present invention also encompassnumerous unique fragments, both longer and shorter than the nucleic acidmolecule sequences set forth in the sequencing listing of the S.agalactiae coding regions, which can be generated by standard cloningmethods. To be unique, a fragment must be of sufficient size todistinguish it from other known nucleic acid sequences, most readilydetermined by comparing any selected S. agalactiae fragment to thenucleotide sequences in computer databases such as GenBank.

Additionally, modifications can be made to the nucleic acid moleculesand polypeptides that are encompassed by the present invention. Forexample, nucleotide substitutions can be made which do not affect thepolypeptide encoded by the nucleic acid, and thus any nucleic acidmolecule which encodes a hyperimmune serum reactive antigen or fragmentsthereof is encompassed by the present invention.

Furthermore, any of the nucleic acid molecules encoding hyperimmuneserum reactive antigens or fragments thereof provided by the presentinvention can be functionally linked, using standard techniques such asstandard cloning techniques, to any desired regulatory sequences,whether a S. agalactiae regulatory sequence or a heterologous regulatorysequence, heterologous leader sequence, heterologous marker sequence ora heterologous coding sequence to create a fusion protein.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA or cRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or produced bychemical synthetic techniques or by a combination thereof. The DNA maybe triple-stranded, double-stranded or single-stranded. Single-strandedDNA may be the coding strand, also known as the sense strand, or it maybe the non-coding strand, also referred to as the anti-sense strand.

The present invention further relates to variants of the herein abovedescribed nucleic acid molecules which encode fragments, analogs andderivatives of the hyperimmune serum reactive antigens and fragmentsthereof having a deducted S. agalactiae amino acid sequence set forth inthe Sequence Listing. A variant of the nucleic acid molecule may be anaturally occurring variant such as a naturally occurring allelicvariant, or it may be a variant that is not known to occur naturally.Such non-naturally occurring variants of the nucleic acid molecule maybe made by mutagenesis techniques, including those applied to nucleicacid molecules, cells or organisms.

Among variants in this regard are variants that differ from theaforementioned nucleic acid molecules by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or additions. Preferred are nucleic acid molecules encoding avariant, analog, derivative or fragment, or a variant, analogue orderivative of a fragment, which have a S. agalactiae sequence as setforth in the Sequence Listing, in which several, a few, 5 to 10, 1 to 5,1 to 3, 2, 1 or no amino acid(s) is substituted, deleted or added, inany combination. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the S. agalactiae polypeptides set forth inthe Sequence Listing. Also especially preferred in this regard areconservative substitutions.

The peptides and fragments according to the present invention alsoinclude modified epitopes wherein preferably one or two of the aminoacids of a given epitope are modified or replaced according to the rulesdisclosed in e.g. {Tourdot, S. et al., 2000}, as well as the nucleicacid sequences encoding such modified epitopes.

It is clear that also epitopes derived from the present epitopes byamino acid exchanges improving, conserving or at least not significantlyimpeding the T cell activating capability of the epitopes are covered bythe epitopes according to the present invention. Therefore the presentepitopes also cover epitopes, which do not contain the original sequenceas derived from S. agalactiae, but trigger the same or preferably animproved T cell response. These epitope are referred to as“heteroclitic”; they need to have a similar or preferably greateraffinity to MHC/HLA molecules, and the need the ability to stimulate theT cell receptors (TCR) directed to the original epitope in a similar orpreferably stronger manner.

Heteroclitic epitopes can be obtained by rational design i.e. takinginto account the contribution of individual residues to binding toMHC/HLA as for instance described by {Rammensee, H. et al., 1999},combined with a systematic exchange of residues potentially interactingwith the TCR and testing the resulting sequences with T cells directedagainst the original epitope. Such a design is possible for a skilledman in the art without much experimentation.

Another possibility includes the screening of peptide libraries with Tcells directed against the original epitope. A preferred way is thepositional scanning of synthetic peptide libraries. Such approaches havebeen described in detail for instance by {Hemmer, B. et al., 1999} andthe references given therein.

As an alternative to epitopes represented by the present derived aminoacid sequences or heteroclitic epitopes, also substances mimicking theseepitopes e.g. “peptidemimetica” or “retro-inverso-peptides” can beapplied.

Another aspect of the design of improved epitopes is their formulationor modification with substances increasing their capacity to stimulate Tcells. These include T helper cell epitopes, lipids or liposomes orpreferred modifications as described in WO 01/78767.

Another way to increase the T cell stimulating capacity of epitopes istheir formulation with immune stimulating substances for instancecytokines or chemokines like interleukin-2, -7, -12, -18, class I and IIinterferons (IFN), especially IFN-gamma, GM-CSF, TNF-alpha, flt3-ligandand others.

As discussed additionally herein regarding nucleic acid molecule assaysof the invention, for instance, nucleic acid molecules of the inventionas discussed above, may be used as a hybridization probe for RNA, cDNAand genomic DNA to isolate full-length cDNAs and genomic clones encodingpolypeptides of the present invention and to isolate cDNA and genomicclones of other genes that have a high sequence similarity to thenucleic acid molecules of the present invention. Such probes generallywill comprise at least 15 bases. Preferably, such probes will have atleast 20, at least 25 or at least 30 bases, and may have at least 50bases. Particularly preferred probes will have at least 30 bases, andwill have 50 bases or less, such as 30, 35, 40, 45, or 50 bases.

For example, the coding region of a nucleic acid molecule of the presentinvention may be isolated by screening a relevant library using theknown DNA sequence to synthesize an oligonucleotide probe. A labeledoligonucleotide having a sequence complementary to that of a gene of thepresent invention is then used to screen a library of cDNA, genomic DNAor mRNA to determine to which members of the library the probehybridizes.

The nucleic acid molecules and polypeptides of the present invention maybe employed as reagents and materials for development of treatments ofand diagnostics for disease, particularly human disease, as furtherdiscussed herein relating to nucleic acid molecule assays, inter alia.

The nucleic acid molecules of the present invention that areoligonucleotides can be used in the processes herein as described, butpreferably for PCR, to determine whether or not the S. agalactiae genesidentified herein in whole or in part are present and/or transcribed ininfected tissue such as blood. It is recognized that such sequences willalso have utility in diagnosis of the stage of infection and type ofinfection the pathogen has attained. For this and other purposes thearrays comprising at least one of the nucleic acids according to thepresent invention as described herein, may be used.

The nucleic acid molecules according to the present invention may beused for the detection of nucleic acid molecules and organisms orsamples containing these nucleic acids. Preferably such detection is fordiagnosis, more preferable for the diagnosis of a disease related orlinked to the present or abundance of S. agalactiae.

Eukaryotes (herein also “individual(s)”), particularly mammals, andespecially humans, infected with S. agalactiae may be identifiable bydetecting any of the nucleic acid molecules according to the presentinvention detected at the DNA level by a variety of techniques.Preferred nucleic acid molecules candidates for distinguishing a S.agalactiae from other organisms can be obtained.

The invention provides a process for diagnosing disease, arising frominfection with S. agalactiae, comprising determining from a sampleisolated or derived from an individual an increased level of expressionof a nucleic acid molecule having the sequence of a nucleic acidmolecule set forth in the Sequence Listing. Expression of nucleic acidmolecules can be measured using any one of the methods well known in theart for the quantitation of nucleic acid molecules, such as, forexample, PCR, RT-PCR, Rnase protection, Northern blotting, otherhybridisation methods and the arrays described herein.

Isolated as used herein means separated “by the hand of man” from itsnatural state; i.e., that, if it occurs in nature, it has been changedor removed from its original environment, or both. For example, anaturally occurring nucleic acid molecule or a polypeptide naturallypresent in a living organism in its natural state is not “isolated,” butthe same nucleic acid molecule or polypeptide separated from thecoexisting materials of its natural state is “isolated”, as the term isemployed herein. As part of or following isolation, such nucleic acidmolecules can be joined to other nucleic acid molecules, such as DNAs,for mutagenesis, to form fusion proteins, and for propagation orexpression in a host, for instance. The isolated nucleic acid molecules,alone or joined to other nucleic acid molecules such as vectors, can beintroduced into host cells, in culture or in whole organisms. Introducedinto host cells in culture or in whole organisms, such DNAs still wouldbe isolated, as the term is used herein, because they would not be intheir naturally occurring form or environment. Similarly, the nucleicacid molecules and polypeptides may occur in a composition, such as amedia formulations, solutions for introduction of nucleic acid moleculesor polypeptides, for example, into cells, compositions or solutions forchemical or enzymatic reactions, for instance, which are not naturallyoccurring compositions, and, therein remain isolated nucleic acidmolecules or polypeptides within the meaning of that term as it isemployed herein.

The nucleic acids according to the present invention may be chemicallysynthesized. Alternatively, the nucleic acids can be isolated from S.agalactiae by methods known to the one skilled in the art.

According to another aspect of the present invention, a comprehensiveset of novel hyperimmune serum reactive antigens and fragments thereofare provided by using the herein described antigen identificationmethod. In a preferred embodiment of the invention, a hyperimmuneserum-reactive antigen comprising an amino acid sequence being encodedby any one of the nucleic acids molecules herein described and fragmentsthereof are provided. In another preferred embodiment of the invention anovel set of hyperimmune serum-reactive antigens which comprises aminoacid sequences selected from a group consisting of the polypeptidesequences as represented in Seq ID No 231, 307, 374-433 and fragmentsthereof are provided. In a further preferred embodiment of the inventionhyperimmune serum-reactive antigens which comprise amino acid sequencesselected from a group consisting of the polypeptide sequences asrepresented in Seq ID No 218, 220, 222-230, 232, 235-242, 244-248,250-253, 256-285, 287-302, 309-317, 320-343, 345-362, 364, 366-373, 434,449-462 and 475-486 and fragments thereof are provided. In a stillpreferred embodiment of the invention hyperimmune serum-reactiveantigens which comprise amino acid sequences selected from a groupconsisting of the polypeptide sequences as represented in Seq ID No 249,303, 308, 318, 344 and fragments thereof are provided. The hyperimmuneserum reactive antigens and fragments thereof as provided in theinvention include any polypeptide set forth in the Sequence Listing aswell as polypeptides which have at least 70% identity to a polypeptideset forth in the Sequence Listing, preferably at least 80% or 85%identity to a polypeptide set forth in the Sequence Listing, and morepreferably at least 90% similarity (more preferably at least 90%identity) to a polypeptide set forth in the Sequence Listing and stillmore preferably at least 95%, 96%, 97%, 98%, 99% or 99.5% similarity(still more preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5%identity) to a polypeptide set forth in the Sequence Listing and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally containing at least 4 amino acids and morepreferably at least 8, still more preferably at least 30, still morepreferably at least 50 amino acids, such as 4, 8, 10, 20, 30, 35, 40, 45or 50 amino acids.

The invention also relates to fragments, analogs, and derivatives ofthese hyperimmune serum reactive antigens and fragments thereof. Theterms “fragment”, “derivative” and “analog” when referring to an antigenwhose amino acid sequence is set forth in the Sequence Listing, means apolypeptide which retains essentially the same or a similar biologicalfunction or activity as such hyperimmune serum reactive antigen andfragment thereof.

The fragment, derivative or analog of a hyperimmune serum reactiveantigen and fragment thereof may be 1) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or 2) one in which one or more of the amino acid residuesincludes a substituent group, or 3) one in which the mature hyperimmuneserum reactive antigen or fragment thereof is fused with anothercompound, such as a compound to increase the half-life of thehyperimmune serum reactive antigen and fragment thereof (for example,polyethylene glycol), or 4) one in which the additional amino acids arefused to the mature hyperimmune serum reactive antigen or fragmentthereof, such as a leader or secretory sequence or a sequence which isemployed for purification of the mature hyperimmune serum reactiveantigen or fragment thereof or a proprotein sequence. Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

The present invention also relates to antigens of different S.agalactiae isolates. Such homologues may easily be isolated based on thenucleic acid and amino acid sequences disclosed herein. There are 9serotypes distinguished to date and the typing is based on serotypespecific antisera. The presence of any antigen can accordingly bedetermined for every serotype. In addition it is possible to determinethe variability of a particular antigen in the various serotypes asdescribed for the S. pyogenes sic gene {Hoe, N. et al., 2001}. Thecontribution of the various serotypes to the different GBS infectionsvaries in the different age groups and geographical regions. It is animportant aspect that the most valuable protective antigens are expectedto be conserved among various clinical strains.

Among the particularly preferred embodiments of the invention in thisregard are the hyperimmune serum reactive antigens set forth in theSequence Listing, variants, analogs, derivatives and fragments thereof,and variants, analogs and derivatives of fragments. Additionally, fusionpolypeptides comprising such hyperimmune serum reactive antigens,variants, analogs, derivatives and fragments thereof, and variants,analogs and derivatives of the fragments are also encompassed by thepresent invention. Such fusion polypeptides and proteins, as well asnucleic acid molecules encoding them, can readily be made using standardtechniques, including standard recombinant techniques for producing andexpression of a recombinant polynucleic acid encoding a fusion protein.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe and Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of any polypeptide set forthin the Sequence Listing, in which several, a few, 5 to 10, 1 to 5, 1 to3, 2, 1 or no amino acid residues are substituted, deleted or added, inany combination. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the polypeptide of the present invention.Also especially preferred in this regard are conservative substitutions.Most highly preferred are polypeptides having an amino acid sequence setforth in the Sequence Listing without substitutions.

The hyperimmune serum reactive antigens and fragments thereof of thepresent invention are preferably provided in an isolated form, andpreferably are purified to homogeneity.

Also among preferred embodiments of the present invention arepolypeptides comprising fragments of the polypeptides having the aminoacid sequence set forth in the Sequence Listing, and fragments ofvariants and derivatives of the polypeptides set forth in the SequenceListing.

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the afore mentioned hyperimmune serum reactive antigen and fragmentthereof, and variants or derivative, analogs, fragments thereof. Suchfragments may be “free-standing”, i.e., not part of or fused to otheramino acids or polypeptides, or they may be comprised within a largerpolypeptide of which they form a part or region. Also preferred in thisaspect of the invention are fragments characterised by structural orfunctional attributes of the polypeptide of the present invention, i.e.fragments that comprise alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta-amphipathic regions, flexibleregions, surface-forming regions, substrate binding regions, and highantigenic index regions of the polypeptide of the present invention, andcombinations of such fragments. Preferred regions are those that mediateactivities of the hyperimmune serum reactive antigens and fragmentsthereof of the present invention. Most highly preferred in this regardare fragments that have a chemical, biological or other activity of thehyperimmune serum reactive antigen and fragments thereof of the presentinvention, including those with a similar activity or an improvedactivity, or with a decreased undesirable activity. Particularlypreferred are fragments comprising receptors or domains of enzymes thatconfer a function essential for viability of S. agalactiae or theability to cause disease in humans. Further preferred polypeptidefragments are those that comprise or contain antigenic or immunogenicdeterminants in an animal, especially in a human.

An antigenic fragment is defined as a fragment of the identifiedantigen, which is for itself antigenic or may be made antigenic whenprovided as a hapten. Therefore, also antigens or antigenic fragmentsshowing one or (for longer fragments) only a few amino acid exchangesare enabled with the present invention, provided that the antigeniccapacities of such fragments with amino acid exchanges are not severelydeteriorated on the exchange(s), i.e., suited for eliciting anappropriate immune response in an individual vaccinated with thisantigen and identified by individual antibody preparations fromindividual sera.

Preferred examples of such fragments of hyperimmune serum-reactiveantigens selected from the group consisting of peptides comprising aminoacid sequences of column “predicted immunogenic aa” and “location ofidentified immunogenic region” of Table 1A, especially peptidescomprising amino acid 4-20, 35-44, 65-70, 73-87, 92-98, 112-137,152-161, 177-186, 193-200, 206-213, 229-255, 282-294, 308-313, 320-326,349-355, 373-384, 388-406, 420-425 and 115-199 of Seq ID No 218; 5-24,35-41, 44-70, 73-89, 103-109, 127-143, 155-161, 185-190, 192-207,212-219, 246-262, 304-336, 372-382, 384-393, 398-407, 412-418, 438-444,1-75, 76-161 and 164-239 of Seq ID No 219; 4-10, 16-58, 60-71, 77-92,100-126, 132-146, 149-164, 166-172, 190-209, 214-220, 223-229, 241-256,297-312, 314-319, 337-343, 351-359, 378-387, 398-418, 421-428, 430-437,440-448, 462-471, 510-519, 525-536, 552-559, 561-568, 573-582, 596-602,608-630, 637-649, 651-665, 681-702, 714-732, 739-745, 757-778, 790-805,807-815, 821-829, 836-842, 846-873, 880-903, 908-914, 916-923, 931-940,943-948, 956-970, 975-986, 996-1015, 1031-1040, 1051-1069, 1072-1095,1114-1119, 1130-1148, 1150-1157, 1169-1176, 1229-1238 and 802-812 of SeqID No 220; 5-12, 14-26, 35-47, 52-67, 72-78, 83-98, 121-141, 152-159,163-183, 186-207, 209-257, 264-277, 282-299, 301-309, 312-318, 324-339,358-368, 372-378, 387-397, 425-431 and 46-291 of Seq ID No 221; 29-38,44-64, 70-76, 78-87, 94-100, 102-112, 119-134, 140-149, 163-173,178-186, 188-194, 207-234, 247-262, 269-290 and 73-92 of Seq ID No 222;10-28, 36-63, 77-87, 103-119, 127-136, 141-169, 171-183, 195-200,207-232, 236-246, 251-265, 268-283, 287-297, 314-322, 335-343, 354-363,384-390, 405-411, 419-436, 443-455, 467-473, 480-513, 518-529, 550-557,565-585, 602-608, 616-625, 632-660, 665-677, 685-701, 726-736, 738-747,752-761, 785-796, 801-813, 838-853, 866-871 and 757-774 of Seq ID No223; 31-38, 61-66, 74-81, 90-115, 123-145, 154-167, 169-179, 182-193,200-206, 238-244, 267-272 and 235-251 of Seq ID No 224; 19-25, 38-54,56-64, 66-72, 74-92, 94-100, 116-129, 143-149, 156-183, 204-232,253-266, 269-275, 294-307 and 241-313 of Seq ID No 225; 5-34, 50-56,60-65, 74-85, 89-97, 108-119, 159-165, 181-199, 209-225, 230-240,245-251, 257-262, 274-282, 300-305 and 64-75 of Seq ID No 226; 5-13,16-21, 27-42, 45-52, 58-66, 74-87, 108-114, 119-131 and 39-51 of Seq IDNo 227; 6-23, 46-54, 59-65, 78-84, 100-120, 128-133, 140-146, 159-165,171-183, 190-204, 224-232, 240-248, 250-259, 274-280, 288-296, 306-315and 267-274 of Seq ID No 228; 5-12, 15-24, 26-36, 42-65, 68-80, 82-104,111-116, 125-144, 159-167, 184-189, 209-218, 235-243, 254-265, 269-283,287-300, 306-316, 318-336, 338-352, 374-392 and 162-174 of Seq ID No229; 30-42, 45-54 and 25-37 of Seq ID No 230; 10-30, 53-59, 86-95,116-130, 132-147, 169-189, 195-201, 212-221, 247-256, 258-265, 278-283,291-298, 310-316, 329-339, 341-352, 360-367, 388-396, 398-411, 416-432,443-452, 460-466, 506-512, 515-521, 542-548 and 419-431 of Seq ID No231; 4-27, 30-53, 60-67, 70-90, 92-151, 159-185, 189-195, 198-210,215-239 and 173-189 of Seq ID No 232; 4-26, 41-54, 71-78, 116-127,140-149, 151-158, 161-175, 190-196, 201-208, 220-226, 240-252, 266-281,298-305, 308-318, 321-329, 344-353, 372-378, 384-405, 418-426, 429-442,457-463, 494-505, 514-522 and 174-188 of Seq ID No 233; 17-25, 27-39,61-67, 81-89, 99-110, 120-131, 133-139, 147-161, 167-172, 179-185,192-198, 203-213, 226-238, 243-258, 261-267, 284-290, 296-307, 311-328,340-352, 356-371 and 239-256 of Seq ID No 234; 8-30, 40-49, 67-80,114-123, 126-142, 152-162, 188-194 and 57-70 of Seq ID No 235; 4-23,28-34, 36-47, 50-61, 76-81, 89-94, 96-104, 112-119, 126-146, 155-181,195-200, 208-214, 220-229, 244-260, 263-276, 282-288, 292-300, 317-323,336-351, 353-359, 363-375, 382-399, 415-432, 444-455, 458-471, 476-481,484-492, 499-517, 522-529, 535-541, 543-568, 572-584, 586-600, 607-617,626-637, 656-675 and 282-297 of Seq ID No 236; 6-24, 30-35, 38-45,63-91, 134-140, 146-160, 167-188, 214-220, 226-234, 244-250, 260-270,286-301, 316-329, 340-371, 429-446, 448-459, 474-481, 485-491, 512-526,537-544, 550-565, 573-583, 596-613, 621-630, 652-658 and 87-97 of Seq IDNo 237; 8-20, 26-48, 56-67, 76-86, 94-109, 115-121, 123-129, 143-160,178-186, 191-198, 201-208, 221-236, 238-244, 260-268 and 237-247 of SeqID No 238; 4-40, 42-57, 73-87, 98-117, 126-135, 150-156, 166-174,196-217, 231-236, 248-258, 276-284, 293-301, 307-313, 339-347, 359-365,375-387, 395-402, 428-440, 445-456, 485-490, 497-505, 535-541, 547-555,610-625, 648-656, 665-671 and 448-528 of Seq ID No 239; 10-18, 39-45,51-61, 80-96, 98-106, 110-115, 158-172, 174-183, 191-200, 220-237,249-255, 274-289, 308-324, 331-341, 372-381, 384-397, 405-414 and322-338 of Seq ID No 240; 30-36, 38-56, 85-108, 134-147, 149-160,163-183, 188-201, 206-211, 219-238, 247-254 and 5-13 of Seq ID No 241;11-40, 98-103, 110-115, 133-145, 151-159, 172-179, 192-201, 204-212,222-228, 235-245, 258-268, 283-296, 298-309, 322-329, 342-351, 354-362,372-378, 385-393, 407-418, 495-516 and 1-148 of Seq ID No 242; 5-19,21-36, 73-94, 112-119, 122-137, 139-145, 152-167, 184-190, 198-204,208-224, 249-265, 267-281, 299-304, 309-317, 326-333, 356-364, 368-374,381-389, 391-414, 419-425, 430-435 and 113-140 of Seq ID No 243; 45-54,59-67, 78-91 and 15-23 of Seq ID No 244; 11-22, 33-47, 52-80, 88-112,124-129 and 6-25 of Seq ID No 245; 26-41, 51-63, 80-89, 93-115, 150-163,187-193, 220-237, 240-249, 286-294, 296-306, 316-329, 345-353, 361-370,407-425, 428-437, 474-482, 484-494, 504-517, 533-541, 549-558, 595-613,616-625, 660-668, 673-685, 711-726, 736-744, 749-761, 787-802, 812-820,825-837, 863-878, 888-896, 901-913, 939-954, 964-972, 977-989,1003-1008, 1016-1022, 1028-1034, 1041-1053, 1059-1074, 1101-1122,420-511 and 581-704 of Seq ID No 246; 18-25, 27-55, 71-83, 89-95,102-113, 120-146, 150-156, 174-185 and 159-175 of Seq ID No 247; 24-30,38-56, 63-68, 87-93, 136-142, 153-164, 183-199, 213-219, 226-234,244-261, 269-278, 283-289, 291-297, 320-328, 330-336, 340-346, 348-356,358-366, 382-387, 401-408, 414-419, 449-455, 468-491, 504-512, 531-537,554-560, 597-608, 621-627, 632-643, 650-662, 667-692, 703-716, 724-737,743-758, 783-794, 800-818, 846-856 and 806-884 of Seq ID No 248; 4-14,21-39, 86-92, 99-107, 121-131, 136-144, 147-154, 158-166, 176-185,193-199, 207-222, 224-230 and 117-136 of Seq ID No 249; 65-76, 85-97,103-109, 115-121, 125-146, 163-169, 196-205, 212-219, 228-237, 241-247,254-262, 269-288, 294-303, 305-313, 328-367, 395-401, 405-412, 418-429,437-447, 481-488, 506-513, 519-524, 530-541, 546-557 and 266-284 of SeqID No 250; 5-14, 37-42, 49-71, 78-92, 97-112, 127-136, 147-154, 156-163,186-198, 216-225, 233-243, 248-253, 295-307, 323-332, 359-366, 368-374,380-398 and 194-223 of Seq ID No 251; 4-11, 33-39, 45-72, 100-113,119-129, 136-144, 169-175, 177-185, 200-208, 210-219, 262-276, 278-297,320-326, 336-344, 347-362, 381-394, 443-453 and 438-454 of Seq ID No252; 4-29, 31-52, 55-61, 95-110, 138-158, 162-171, 179-187, 202-229,239-248, 251-256, 262-267, 269-285, 304-310, 351-360, 362-368, 381-388,415-428, 435-440, 448-458 and 161-178 of Seq ID No 253; 4-17, 19-28,32-43, 47-59, 89-110, 112-126, 128-134, 140-148, 152-161, 169-184,191-204, 230-235, 255-264, 328-338, 341-347, 401-409, 413-419, 433-441,449-458, 463-468, 476-482, 486-492, 500-506, 529-545 and 305-381 of SeqID No 254; 10-29, 38-45, 53-61, 134-145, 152-160, 163-170, 202-208,219-229, 248-258, 266-275, 282-288, 315-320, 328-334, 377-385, 392-402,418-424, 447-453, 460-471, 479-487, 491-497, 500-507, 531-537, 581-594,615-623, 629-635, 644-652, 659-666, 668-678, 710-717, 719-728, 736-741,747-760, 766-773, 784-789, 794-800, 805-817, 855-861, 866-887 and698-715 of Seq ID No 255; 16-26, 29-37, 44-58, 62-68, 74-80, 88-95,97-120, 125-144, 165-196 and 58-72 of Seq ID No 256; 14-21, 23-46,49-60, 63-74, 78-92, 96-103, 117-129, 134-161, 169-211, 217-231,239-248, 252-281, 292-299, 313-343 and 243-257 of Seq ID No 257; 11-27,46-52, 67-72, 76-84, 91-112, 116-153, 160-175, 187-196, 202-211, 213-220and 43-76 of Seq ID No 258; 5-29, 37-56, 78-86, 108-118, 152-161 and120-130 of Seq ID No 259; 8-14, 19-41, 52-66, 75-82, 87-92, 106-121,127-133, 136-143, 158-175, 180-187, 196-204, 221-228, 239-245, 259-265,291-306, 318-323, 328-340, 352-358, 361-368, 375-381, 391-399, 411-418,431-442, 446-455, 484-496, 498-510, 527-533, 541-549, 558-565, 575-585,587-594, 644-655, 661-668, 671-677 and 184-196 of Seq ID No 260; 4-22,29-38, 55-62, 75-81, 102-107, 110-134, 143-150, 161-167, 172-179,191-215, 223-233, 241-247, 251-264, 266-272, 288-309, 340-352, 354-366,394-402, 414-438 and 198-218 of Seq ID No 261; 24-44, 49-70, 80-91,105-118, 128-136, 140-154 and 77-92 of Seq ID No 262; 5-22, 31-36,41-47, 67-74, 83-90, 105-122, 135-143, 160-167 and 118-129 of Seq ID No263; 4-25, 33-73, 81-93, 96-106, 114-120, 122-128, 130-172, 179-208,210-241, 251-283, 296-301 and 92-100 of Seq ID No 264; 14-24, 29-38,43-50, 52-72, 86-97, 101-107, 110-125, 127-141, 145-157, 168-175,177-184, 186-195, 205-226, 238-250, 255-261, 284-290, 293-304, 307-314,316-323, 325-356, 363-371, 383-390, 405-415, 423-432, 442-454, 466-485,502-511, 519-527, 535-556, 558-565, 569-574, 612-634, 641-655, 672-686,698-709, 715-722, 724-732, 743-753, 760-769, 783-792, 818-825, 830-839,842-849, 884-896, 905-918, 926-940, 957-969, 979-1007, 1015-1021,1049-1057 and 336-349 of Seq ID No 265; 6-16, 26-31, 33-39, 62-73,75-85, 87-100, 113-123, 127-152, 157-164, 168-181, 191-198, 208-214,219-226, 233-254, 259-266, 286-329 and 181-195 of Seq ID No 266; 4-13,32-39, 53-76, 99-108, 110-116, 124-135, 137-146, 149-157, 162-174,182-190, 207-231, 242-253, 255-264, 274-283, 291-323, 334-345, 351-360,375-388, 418-425, 456-474, 486-492, 508-517, 520-536, 547-560, 562-577,31-45 and 419-443 of Seq ID No 267; 15-26, 30-37, 42-49, 58-90, 93-99,128-134, 147-154, 174-179, 190-197, 199-205, 221-230, 262-274, 277-287,300-314, 327-333, 343-351, 359-377, 388-396, 408-413, 416-425, 431-446and 246-256 of Seq ID No 268; 5-26, 34-42, 47-54, 61-67, 71-104,107-115, 131-138, 144-153, 157-189, 196-202, 204-210, 228-245, 288-309,316-329, 332-341, 379-386, 393-399, 404-412, 414-421, 457-468, 483-489,500-506, 508-517, 523-534, 543-557, 565-580, 587-605, 609-617, 619-627,631-636, 640-646, 662-668, 675-682, 705-710, 716-723, 727-732, 750-758,784-789, 795-809, 869-874, 14-138, 166-286, 372-503, 674-696 and 754-859of Seq ID No 269; 5-17, 32-38, 40-47, 80-89, 113-119, 125-137, 140-154,157-163, 170-177, 185-199, 213-225, 228-236, 242-248, 277-290, 292-305,323-333, 347-353, 364-370, 385-394, 399-406, 423-433, 441-451, 462-474,477-487 and 116-124 of Seq ID No 270; 7-16, 18-30, 32-49, 53-61, 63-85,95-101, 105-115, 119-134, 143-150, 159-178, 185-202, 212-229, 236-250,254-265, 268-294 and 63-72 of Seq ID No 271; 4-12, 19-47, 73-81, 97-103,153-169, 188-198, 207-213, 217-223, 236-242, 255-265, 270-278, 298-305,309-317, 335-347, 354-363, 373-394, 419-424, 442-465, 486-492, 500-507,542-549, 551-558, 560-572, 580-589, 607-614, 617-623, 647-653, 666-676,694-704, 706-714, 748-754, 765-772, 786-792, 795-806 and 358-370 of SeqID No 272; 18-28, 30-38, 40-46, 49-55, 69-78, 82-98, 104-134, 147-153,180-190, 196-202, 218-236, 244-261, 266-273, 275-286, 290-295, 301-314,378-387, 390-395, 427-434 and 290-305 of Seq ID No 273; 4-13, 20-31,39-51, 54-61, 69-84, 87-105, 117-124 and 108-125 of Seq ID No 274;24-34, 43-54, 56-66, 68-79 and 50-69 of Seq ID No 275; 5-43, 71-77,102-131, 141-148, 150-156, 159-186, 191-207, 209-234, 255-268, 280-286,293-299, 317-323, 350-357, 363-372, 391-397, 406-418, 428-435, 455-465,484-497, 499-505, 525-531, 575-582, 593-607, 621-633, 638-649, 655-673,684-698, 711-725, 736-741, 743-752, 759-769, 781-793, 813-831, 843-853,894-905, 908-916, 929-946, 953-963, 970-978, 1001-1007, 1011-1033,165-178 and 818-974 of Seq ID No 276; 16-44, 63-86, 98-108, 185-191,222-237, 261-274, 282-294, 335-345, 349-362, 374-384, 409-420, 424-430,440-447, 453-460, 465-473, 475-504, 522-534, 538-551, 554-560, 567-582,598-607, 611-619, 627-640, 643-653, 655-661, 669-680, 684-690, 701-707,715-731, 744-750, 756-763, 768-804, 829-837, 845-853, 855-879, 884-890,910-928, 77-90, 144-212, 279-355, 434-536, 782-810 and 875-902 of Seq IDNo 277; 4-22, 29-41, 45-51, 53-66, 70-77, 86-95, 98-104, 106-124,129-135, 142-151, 153-161, 169-176, 228-251, 284-299, 331-337, 339-370,380-387, 393-398, 406-411, 423-433, 440-452, 461-469, 488-498, 501-516,523-530, 532-559, 562-567, 570-602, 612-628, 630-645, 649-659, 666-672,677-696, 714-723, 727-747 and 212-227 of Seq ID No 278; 4-9, 17-31,35-41, 56-61, 66-75, 81-87, 90-124, 133-138, 149-163, 173-192, 213-219,221-262, 265-275, 277-282, 292-298, 301-307, 333-346, 353-363, 371-378,419-430, 435-448, 456-469, 551-570, 583-599, 603-612 and 275-291 of SeqID No 279; 28-34, 53-58, 72-81, 100-128, 145-154, 159-168, 172-189,217-225, 227-249, 256-263, 299-309, 322-330, 361-379, 381-388, 392-401,404-417, 425-436, 440-446, 451-464, 469-487, 502-511, 543-551, 559-564,595-601, 606-612, 615-626, 633-642, 644-650, 664-670, 674-684, 692-701,715-723, 726-734, 749-756, 763-771, 781-787, 810-843, 860-869, 882-889,907-917, 931-936, 941-948, 951-958, 964-971, 976-993, 1039-1049,1051-1065, 1092-1121, 1126-1132, 1145-1151, 1158-1173, 1181-1192,1194-1208, 1218-1223, 1229-1243, 1249-1254, 1265-1279, 1287-1297,1303-1320, 1334-1341, 1343-1358, 1372-1382, 1406-1417, 1419-1425,1428-1434, 1441-1448, 1460-1473, 1494-1504, 1509-1514, 1529-1550,654-669 and 1400-1483 of Seq ID No 280; 10-16, 20-25, 58-65, 97-109,118-132, 134-146, 148-155, 186-195, 226-233, 244-262, 275-284, 295-310,317-322, 330-339, 345-351, 366-375, 392-403, 408-415, 423-430, 435-444,446-457, 467-479, 486-499, 503-510, 525-537, 540-585, 602-612, 614-623,625-634, 639-645, 650-669, 700-707, 717-724, 727-739, 205-230 and733-754 of Seq ID No 281; 5-22, 37-43, 72-81, 105-113, 128-133, 148-160,188-194, 204-230, 238-245, 251-257 and 194-213 of Seq ID No 282; 16-21,35-41, 56-72, 74-92, 103-109 and 62-68 of Seq ID No 283; 4-15, 17-82,90-104, 107-159, 163-170, 188-221, 234-245, 252-265 and 220-235 of SeqID No 284; 16-22, 36-46, 61-75, 92-107, 113-121, 139-145, 148-160 and30-42 of Seq ID No 285; 4-12, 20-26, 43-49, 55-62, 66-78, 121-127,135-141, 146-161, 164-170, 178-189, 196-205, 233-238, 269-279, 288-318,325-332, 381-386, 400-407 and 328-346 of Seq ID No 286; 5-12, 31-49,57-63, 69-79, 89-97, 99-114, 116-127, 134-142, 147-154, 160-173,185-193, 199-204, 211-222, 229-236, 243-249, 256-274 and 58-68 of Seq IDNo 287; 10-20, 28-34, 39-53, 68-79, 84-90, 99-106 and 73-79 of Seq ID No288; 14-37, 45-50, 61-66, 77-82, 93-98, 109-114, 125-130, 141-146,157-162, 173-178, 189-194, 205-210, 221-226, 237-242, 253-258, 269-274,285-290, 301-306, 316-332, 349-359, 371-378, 385-406, 34-307 and 312-385of Seq ID No 289; 4-10, 17-38, 50-85, 93-99, 109-116, 128-185, 189-197,199-210, 223-256, 263-287, 289-312, 327-337, 371-386, 389-394, 406-419,424-432, 438-450, 458-463, 475-502, 507-513, 519-526, 535-542, 550-567and 361-376 of Seq ID No 290; 10-39, 42-93, 100-144, 155-176, 178-224,230-244, 246-255, 273-282, 292-301, 308-325, 332-351, 356-361, 368-379,386-393, 400-421 and 138-155 of Seq ID No 291; 5-11, 17-34, 40-45,50-55, 72-80, 101-123, 145-151, 164-172, 182-187, 189-195, 208-218,220-241, 243-252, 255-270, 325-331, 365-371, 391-398, 402-418, 422-428,430-435, 443-452, 463-469, 476-484, 486-494, 503-509, 529-553, 560-565,570-590, 608-614, 619-627, 654-661, 744-750, 772-780, 784-790, 806-816,836-853, 876-885, 912-918, 926-933, 961-975, 980-987, 996-1006,1016-1028, 1043-1053, 1057-1062, 994-1003 and 1033-1056 of Seq ID No292; 17-45, 64-71, 73-81, 99-109, 186-192, 223-238, 262-275, 283-295,336-346, 350-363, 375-385, 410-421, 425-431, 441-448, 454-463, 468-474,476-512, 523-537, 539-552, 568-583, 599-608, 612-620, 628-641, 644-654,656-662, 670-681, 685-695, 702-708, 716-723, 725-735, 757-764, 769-798,800-806, 808-816, 826-840, 846-854, 856-862, 874-881, 885-902, 907-928,274-350 and 443-513 of Seq ID No 293; 4-22, 29-41, 45-51, 53-61, 70-76,85-92, 99-104, 111-122, 134-140, 142-154, 163-174, 224-232, 255-265,273-279, 283-297, 330-335, 337-348, 356-367, 373-385, 391-396, 421-431,442-455, 475-485, 493-505, 526-538, 544-561, 587-599, 605-620, 622-651,662-670, 675-681, 687-692, 697-712, 714-735 and 252-262 of Seq ID No294; 4-12, 15-35, 40-46, 50-59, 67-94, 110-128, 143-169, 182-188,207-215, 218-228, 238-250 and 74-90 of Seq ID No 295; 9-18, 42-58,78-85, 88-95, 97-106, 115-122, 128-134, 140-145, 154-181, 186-202,204-223, 261-267, 269-278, 284-293, 300-336, 358-368 and 12-29 of Seq IDNo 296; 7-34, 46-53, 62-72, 82-88, 100-105, 111-117, 132-137, 144-160,166-180, 183-189, 209-221, 231-236, 246-253, 268-282, 286-293, 323-336,364-372, 378-392, 422-433 and 388-405 of Seq ID No 297; 21-27, 34-50,72-77, 80-95, 164-177, 192-198, 202-220, 226-236, 239-247, 270-279,285-292, 315-320, 327-334, 348-355, 364-371, 388-397, 453-476, 488-497,534-545, 556-576, 582-588, 601-607, 609-616, 642-662, 674-681, 687-697,709-715, 721-727, 741-755 and 621-739 of Seq ID No 298; 4-14, 16-77,79-109 and 25-99 of Seq ID No 299; 4-9, 17-23, 30-37, 44-55, 65-72,77-93, 102-121, 123-132, 146-153 and 17-29 of Seq ID No 300; 4-18,25-41, 52-60, 83-92, 104-112, 117-123, 149-155, 159-167, 170-192,201-210, 220-227, 245-250 and 124-137 of Seq ID No 301; 8-25, 50-55,89-95, 138-143, 148-153, 159-169, 173-179, 223-238, 262-268, 288-295,297-308, 325-335, 403-409, 411-417, 432-446, 463-475, 492-501, 524-530,542-548, 561-574, 576-593, 604-609, 612-622, 637-654, 665-672, 678-685,720-725, 731-739, 762-767, 777-783, 820-838, 851-865, 901-908, 913-920,958-970, 1000-1006, 1009-1015, 1020-1026, 1043-1052, 1055-1061, 1-128,252-341, 771-793 and 1043-1058 of Seq ID No 302; 16-26, 33-46 and 64-76of Seq ID No 303; 4-27, 69-77, 79-101, 117-123, 126-142, 155-161,171-186, 200-206, 213-231, 233-244, 267-273, 313-329, 335-344, 347-370,374-379, 399-408, 422-443, 445-453, 461-468, 476-482, 518-534, 544-553,556-567, 578-595, 601-620, 626-636, 646-658, 666-681, 715-721, 762-768,778-785, 789-803, 809-819, 22-108, 153-318, 391-527 and 638-757 of SeqID No 304; 6-21, 32-43, 62-92, 104-123, 135-141, 145-152, 199-216,218-226, 237-247, 260-269, 274-283, 297-303, 1-72 and 127-211 of Seq IDNo 305; 6-26, 50-56, 83-89, 108-114, 123-131, 172-181, 194-200, 221-238,241-247, 251-259, 263-271, 284-292, 304-319, 321-335, 353-358, 384-391,408-417, 424-430, 442-448, 459-466, 487-500, 514-528, 541-556, 572-578,595-601, 605-613, 620-631, 635-648, 660-670, 673-679, 686-693, 702-708,716-725, 730-735, 749-755, 770-777, 805-811, 831-837, 843-851, 854-860,863-869, 895-901, 904-914, 922-929, 933-938, 947-952, 956-963,1000-1005, 1008-1014, 1021-1030, 1097-1103, 1120-1130, 1132-1140, 1-213,269-592 and 992-1120 of Seq ID No 306; 9-16, 33-39, 47-59, 65-79, 81-95,103-108, 115-123, 138-148, 163-171, 176-185, 191-196, 205-211, 213-221,224-256, 261-276, 294-302, 357-363, 384-390, 95-111 and 161-189 of SeqID No 307; 21-27, 35-45, 70-76, 92-105, 129-143, 145-155, 161-166,170-191, 204-211, 214-231, 234-246, 249-255, 259-275 and 1-18 of Seq IDNo 308; 21-35, 45-53, 56-64, 69-97 and 1-16 of Seq ID No 309; 25-33,41-47, 61-68, 86-101, 106-114, 116-129, 134-142, 144-156, 163-176,181-190, 228-251, 255-261, 276-292, 295-305, 334-357, 368-380, 395-410,424-429, 454-460, 469-482, 510-516, 518-527, 531-546, 558-570, 579-606,628-636, 638-645, 651-656, 668-674, 691-698, 717-734, 742-754, 765-770,792-797, 827-835, 847-859, 874-881, 903-909, 926-933, 942-961, 964-977,989-1004, 1010-1028, 1031-1047, 1057-1075, 1081-1095, 1108-1117,1138-1144, 1182-1189, 1193-1206, 1220-1229, 1239-1246, 1257-1267,1271-1279, 1284-1301, 1312-1320, 1329-1335, 1341-1347, 1358-1371,1399-1404, 1417-1426, 1458-1463, 1468-1476, 1478-1485, 1493-1506,1535-1541, 1559-1574, 1583-1590, 1595-1601, 1603-1611, 1622-1628,1634-1644, 1671-1685, 1689-1696, 1715-1720, 1734-1746, 1766-1775,1801-1806, 1838-1844, 1858-1871, 1910-1917, 1948-1955, 1960-1974,2000-2015, 2019-2036, 2041-2063, 748-847 and 1381-1391 of Seq ID No 310;5-12, 18-24, 27-53, 56-63, 96-113, 119-124, 131-136, 157-163, 203-209,215-223, 233-246, 264-273, 311-316, 380-389, 393-399, 425-433, 445-450,457-462, 464-470, 475-482, 507-513, 527-535, 542-548, 550-565, 591-602,607-613, 627-642, 644-664, 673-712, 714-732, 739-764, 769-782, 812-818,826-838, 848-854, 860-871, 892-906, 930-938, 940-954, 957-973, 990-998,1002-1021, 1024-1033, 1037-1042, 1050-1060, 1077-1083, 1085-1092,1100-1129, 1144-1161, 1169-1175, 1178-1189, 1192-1198, 1201-1207,1211-1221, 1229-1239, 1250-1270, 1278-1292, 1294-1300, 1314-1335,1344-1352, 1360-1374, 1394-1405, 1407-1414, 1416-1424, 1432-1452,1456-1462, 1474-1497, 1500-1510, 1516-1522, 1534-1542, 1550-1559,1584-1603, 1608-1627, 187-273 and 306-441 of Seq ID No 311; 70-80,90-97, 118-125, 128-140, 142-148, 154-162, 189-202, 214-222, 224-232,254-260, 275-313, 317-332, 355-360, 392-398, 425-432, 448-456, 464-470,476-482, 491-505, 521-528, 533-546, 560-567, 592-597, 605-614, 618-626,637-644, 646-653, 660-666, 677-691 and 207-227 of Seq ID No 312; 5-19,26-34, 37-55, 57-66, 69-83, 86-102, 115-134, 138-143, 154-172, 178-195,209-246, 251-257, 290-302, 306-311 and 256-266 of Seq ID No 313; 10-20,22-28, 35-57, 72-79, 87-103, 108-128, 130-144, 158-171, 190-198,225-242, 274-291, 301-315, 317-324, 374-385 and 353-365 of Seq ID No314; 4-9, 17-30, 34-54, 59-66, 73-94, 118-130, 135-150, 158-171,189-198, 219-239, 269-275, 283-301, 89-106 and 176-193 of Seq ID No 315;14-20, 22-74, 77-86, 89-99, 104-109, 126-135, 154-165, 181-195, 197-212,216-224, 264-275 and 107-118 of Seq ID No 316; 4-18, 21-38, 63-72,101-109, 156-162, 165-179, 183-192, 195-210, 212-218, 230-239, 241-256,278-290, 299-311, 313-322, 332-341, 348-366, 386-401, 420-426, 435-450,455-460, 468-479, 491-498, 510-518, 532-538, 545-552, 557-563, 567-573,586-595, 599-609, 620-626, 628-636, 652-657, 665-681 and 1-198 of Seq IDNo 317; 4-10, 16-38, 51-68, 73-79, 94-115, 120-125, 132-178, 201-208,216-223, 238-266, 269-295, 297-304, 337-342, 347-356, 374-401, 403-422,440-447, 478-504, 510-516, 519-530, 537-544 and 191-206 of Seq ID No318; 12-40, 42-48, 66-71, 77-86, 95-102, 113-120, 129-137, 141-148,155-174, 208-214, 218-225, 234-240, 256-267, 275-283, 300-306, 313-321,343-350, 359-367, 370-383, 398-405, 432-439, 443-461, 492-508, 516-526,528-535 and 370-478 of Seq ID No 319; 6-14, 20-37, 56-62, 90-95, 97-113,118-125, 140-145, 161-170, 183-202, 237-244, 275-284, 286-305, 309-316,333-359, 373-401, 405-412 and 176-187 of Seq ID No 320; 33-44, 50-55,59-80, 86-101, 129-139, 147-153, 157-163, 171-176, 189-201, 203-224,239-245, 257-262, 281-287, 290-297, 304-320, 322-331, 334-350, 372-390,396-401, 71-88 and 353-372 of Seq ID No 321; 5-11, 15-24, 26-33, 40-47,75-88, 95-103, 105-112 and 17-30 of Seq ID No 322; 5-11, 16-39, 46-54,62-82, 100-107, 111-124, 126-150, 154-165, 167-183, 204-238, 245-295,301-313, 316-335 and 8-16 of Seq ID No 323; 4-19, 34-48, 69-74, 79-107,115-127, 129-135, 143-153, 160-169, 171-182 and 142-153 of Seq ID No324; 4-30, 65-74, 82-106, 110-120, 124-132, 135-140, 146-175, 179-184,190-196, 217-223, 228-233, 250-267, 275-292, 303-315, 322-332 and174-186 of Seq ID No 325; 9-16, 29-41, 47-57, 68-84, 87-109, 113-119,162-180, 186-193, 195-201, 203-208, 218-230, 234-243, 265-271, 281-292,305-312, 323-332, 341-347, 349-363, 368-374, 383-390, 396-410, 434-440,446-452, 455-464, 466-473, 515-522, 529-542, 565-570, 589-600, 602-613,618-623, 637-644, 1019-1027, 1238-1244, 1258-1264, 1268-1276, 1281-1292,1296-1302 and 883-936 of Seq ID No 326; 10-17, 23-32, 39-44, 54-72,75-81, 88-111, 138-154, 160-167, 178-185, 201-210, 236-252, 327-334,336-342, 366-376, 388-400, 410-430, 472-482, 493-526, 552-558, 586-592,598-603, 612-621, 630-635, 641-660 and 384-393 of Seq ID No 327; 4-22,24-39, 50-59, 73-84, 100-105, 111-117, 130-138, 155-161, 173-178,182-189, 205-215, 266-284, 308-313, 321-328, 330-337, 346-363, 368-374,388-395, 397-405, 426-434, 453-459, 482-492, 501-507, 509-515, 518-523,527-544, 559-590, 598-612, 614-629, 646-659, 663-684, 686-694, 698-721and 445-461 of Seq ID No 328; 14-22, 27-33 and 3-17 of Seq ID No 329;29-41, 66-73, 81-87, 90-108, 140-146, 150-159, 165-184, 186-196,216-226, 230-238, 247-253, 261-269 and 126-140 of Seq ID No 330; 5-12,16-25, 27-33, 36-45, 60-68, 83-88, 103-126 and 86-101 of Seq ID No 331;14-23, 36-47, 56-66, 84-89, 94-105, 111-127, 140-153, 160-174, 176-183,189-203, 219-225, 231-237, 250-257 and 194-227 of Seq ID No 332; 4-25,54-60, 64-71, 73-82, 89-106, 117-124, 157-169, 183-188, 199-210,221-232, 236-244, 255-264 and 58-98 of Seq ID No 333; 13-19, 26-36,41-53, 55-71, 77-84, 86-108, 114-135, 157-172, 177-183, 187-194,208-213, 218-226, 110-125 and 156-170 of Seq ID No 334; 5-24, 63-69,77-85, 94-112, 120-137, 140-146, 152-159, 166-172, 179-187, 193-199,206-212, 222-228, 234-240, 244-252, 257-264, 270-289, 298-309, 316-328,337-348, 363-375, 1-56 and 340-352 of Seq ID No 335; 18-39, 42-71,78-120, 124-144, 152-173, 179-189, 199-209, 213-222, 228-258, 269-304,329-361, 364-372, 374-389, 396-441 and 313-327 of Seq ID No 336; 19-25,91-98, 108-120, 156-162, 168-174, 191-204, 211-216, 232-266, 272-278,286-308, 316-321, 327-333, 344-355, 358-364, 384-391, 395-428, 464-476,487-495, 497-511, 544-561, 563-573, 575-582, 588-594, 10-25 and 322-338of Seq ID No 337; 14-26, 32-49, 51-57, 59-72, 80-91, 102-112, 119-125,147-161, 164-173, 175-183, 188-213, 217-222, 246-254, 260-276, 282-303,308-318, 321-328, 333-350, 352-359, 371-378, 392-401, 407-414, 416-443,448-463, 471-484, 490-497, 501-514, 519-527, 539-551, 557-570, 578-590,592-598, 600-610, 618-629, 633-647, 654-667, 676-689, 702-709, 718-726,728-737, 741-760, 764-780, 786-795, 808-826, 836-842, 845-852, 865-874,881-887, 931-945, 949-957, 968-974, 979-986, 1003-1009, 1023-1029 and90-103 of Seq ID No 338; 11-16, 37-56, 60-66, 69-77, 80-88, 93-106,117-139, 166-171 and 72-90 of Seq ID No 339; 59-84, 123-133, 145-150,161-167, 178-189 and 115-128 of Seq ID No 340; 15-33, 39-46, 52-64,74-87, 108-124, 127-144, 150-156, 173-179, 184-194, 201-208, 219-236,243-269, 272-295, 302-309, 343-349, 356-361, 370-379, 405-411, 414-423,430-451, 457-464, 466-475, 477-483, 496-502, 507-522, 541-548, 557-563,571-577, 579-585, 590-605, 626-642, 650-662, 671-691, 704-710, 751-769,775-781, 786-791, 794-829, 851-858, 868-878, 884-904, 913-919, 931-939and 132-142 of Seq ID No 341; 33-58, 64-71, 74-80, 83-88, 96-120,122-139, 146-157, 167-177, 207-213, 220-225, 236-242, 264-279, 300-305,326-336, 340-347, 350-360, 97-115 and 199-211 of Seq ID No 342; 4-26,43-57, 70-99, 102-117, 121-133, 142-148, 151-168, 170-183, 192-220,235-249, 258-279 and 30-41 of Seq ID No 343; 34-42, 48-58, 70-94,110-130, 154-160, 164-172, 178-183, 195-203, 211-222, 229-250, 256-261,274-284, 286-292, 312-323 and 222-233 of Seq ID No 344; 4-9, 15-36,38-45, 49-74, 78-88, 100-112, 136-191, 211-220, 226-233, 239-246,254-274, 287-307, 316-322, 342-353, 356-366, 373-378, 384-393, 405-431,449-457, 459-468, 487-511, 515-524, 529-541, 544-552, 562-568, 571-576and 208-280 of Seq ID No 345; 10-27, 31-37, 39-54, 71-108, 124-143 and2-107 of Seq ID No 346; 16-27, 38-57, 64-70, 90-102, 104-113, 116-137,160-166 and 1-80 of Seq ID No 347; 13-21, 31-36, 56-67, 127-136,153-171, 173-180, 184-200, 214-222, 225-231, 239-263, 267-273 and135-159 of Seq ID No 348; 12-27, 31-51, 68-74, 77-87, 94-101, 108-114,117-123, 127-134, 138-168, 173-196, 201-207, 212-217, 227-237, 247-257,264-280 and 205-223 of Seq ID No 349; 17-22, 25-54, 70-76, 92-100 and98-110 of Seq ID No 350; 7-29, 40-50, 60-67, 87-96, 105-111, 119-164,172-199, 206-212, 220-227, 237-259, 272-279, 282-293, 295-309, 313-319,321-328, 345-363, 376-386 and 159-176 of Seq ID No 351; 4-19, 24-30,36-43, 50-68, 71-89, 93-106, 141-152, 154-172, 179-197, 199-215,229-239, 246-252, 255-263, 281-298, 319-325, 329-356, 358-368, 374-390,397-409, 420-429, 432-444, 450-456, 459-475, 483-494, 496-502, 520-528,532-556 and 362-377 of Seq ID No 352; 18-25, 40-62, 77-85, 91-97,105-116, 123-133, 139-184, 189-197 and 122-140 of Seq ID No 353; 4-49,52-58, 62-70, 79-105, 109-133, 142-150, 163-168, 206-214, 220-228,233-240, 243-254, 274-281, 303-311, 327-338, 357-373, 378-396, 403-413,420-436, 441-453, 461-467, 475-481, 484-498, 506-512, 514-521, 523-529,562-579, 589-595, 598-603, 615-648, 714-722, 728-742, 749-758, 777-792,795-807 and 643-658 of Seq ID No 354; 8-27, 37-48, 51-56, 72-79, 87-106,120-138, 140-147, 167-176, 187-197, 205-216, 222-229, 234-239, 243-249,277-288, 292-315, 334-343, 347-353, 363-391, 398-404, 430-447, 461-467,478-492, 498-507 and 456-470 of Seq ID No 355; 5-12, 18-24, 59-69,80-93, 95-109, 119-125, 130-137, 139-147, 158-163, 168-176, 182-202,206-215, 222-239, 241-249, 267-277, 291-298, 311-318, 321-327, 338-344,348-355, 373-386, 393-406, 411-417, 434-443, 446-465, 473-484, 514-521,532-553, 584-594 and 221-237 of Seq ID No 356; 4-14, 27-34, 50-58,63-72, 79-106, 109-114, 121-142, 146-154, 161-167, 169-175, 178-201,223-238, 249-254, 259-264, 278-292, 294-312, 319-330 and 167-191 of SeqID No 357; 7-28, 36-42, 50-61, 63-80, 122-152, 161-174, 176-191 and140-190 of Seq ID No 358; 20-57, 59-65, 70-78, 86-102, 119-133, 142-161,163-173, 177-188, 192-202, 204-220, 222-236, 240-253, 279-319, 326-331,337-383, 390-399, 406-412, 420-427, 431-438 and 381-395 of Seq ID No359; 13-18, 28-34, 37-43, 50-59, 75-81, 83-97, 105-121, 139-147,200-206, 209-227, 231-247, 260-271, 318-327, 366-381, 388-394, 399-406and 182-201 of Seq ID No 360; 6-29, 37-43, 51-56, 70-77, 82-102,110-119, 127-143, 178-190, 201-209, 216-243, 261-269, 281-292, 305-313,327-339, 341-354, 356-373, 391-397, 423-429, 438-445, 450-478 and 21-314of Seq ID No 361; 4-12, 15-21, 32-41, 59-76, 80-89, 96-104 and 90-103 ofSeq ID No 362; 9-28, 30-41, 44-54, 69-74, 77-82, 90-97, 104-123,125-135, 149-155, 164-173, 177-184, 217-226, 230-235, 238-244, 258-272,282-297, 300-305, 309-315, 317-322, 327-336, 348-362, 368-374, 380-387,400-411, 414-424, 451-458, 460-466, 483-494, 497-503, 506-511, 521-528,540-553, 569-587, 598-606, 628-642, 661-681, 688-700, 718-733, 740-749,752-764, 769-783, 823-834, 848-854, 862-872, 878-884, 886-898, 915-920,938-951, 954-961, 963-972, 982-989, 996-1003, 1010-1016, 1021-1032,1038-1044, 1047-1057, 1060-1070, 1079-1088, 1094-1102, 1117-1127,1129-1135, 1142-1153, 1158-1204, 1212-1229, 1234-1263, 1269-1277,1308-1313, 1327-1338, 1344-1376, 1400-1415, 1436-1443, 1448-1458,1497-1504, 1511-1522, 1544-1566, 3-82 and 509-576 of Seq ID No 363;8-36, 40-64, 71-79, 88-94, 102-109, 118-127, 138-148, 151-159, 163-174,192-198, 200-206, 220-233, 268-273, 290-301, 304-309, 316-323, 331-349,378-391, 414-420, 427-437, 455-475, 494-510, 541-547, 549-555, 616-640,1-60, 55-139, 212-308, 386-458 and 458-624 of Seq ID No 364; 16-31,35-42, 70-77, 91-101, 120-130, 132-140, 143-153, 185-190, 195-202,215-222, 228-238, 241-251, 257-264, 268-277, 288-302, 312-324, 326-333,341-348, 364-382, 415-429, 438-454, 458-466, 491-499, 501-521 and273-281 of Seq ID No 365; 8-14, 32-57, 74-149, 155-177, 179-212,221-266, 271-296, 304-324, 329-346, 349-359, 368-401, 413-419, 426-454,465-478, 493-510 and 466-490 of Seq ID No 366; 22-28, 33-51, 64-89,96-119, 126-132, 138-146, 152-159, 161-169, 172-179, 193-198, 205-211,221-231, 235-254, 273-280, 297-303, 312-320, 328-346, 351-373, 378-384,391-398, 448-454, 460-468, 470-481, 516-558, 574-593, 597-602, 613-623,626-646, 649-656, 668-673, 675-683, 696-708, 715-722, 724-739, 745-751,759-777, 780-804, 816-822 and 102-113 of Seq ID No 367; 12-28, 41-91,98-107, 112-120, 125-131, 151-193, 215-221, 240-250, 263-280 and 128-138of Seq ID No 368; 16-24, 32-38, 46-62, 68-81, 90-105, 127-133, 144-150,160-166, 178-184, 186-202, 210-219, 232-240, 252-258, 264-273, 293-324,337-344, 349-357, 360-369, 385-398, 410-416, 419-427, 441-449, 458-476,508-515, 523-539, 544-549, 562-569, 571-579, 96-109 and 127-139 of SeqID No 369; 19-25, 28-34, 56-61, 85-97, 110-116 and 39-53 of Seq ID No370; 4-37, 41-50, 62-72, 91-97, 99-109, 114-125, 136-141, 149-158,160-166, 201-215 and 27-225 of Seq ID No 371; 15-31, 44-51, 96-105,122-130, 149-157, 162-168, 178-183, 185-192, 198-204, 206-213, 221-234,239-245, 248-255, 257-266, 289-335, 349-357, 415-422, 425-441, 448-454,462-468 and 463-481 of Seq ID No 372; 5-31, 39-55, 63-72, 76-99,106-155, 160-177, 179-199, 207-217, 223-240, 245-255, 261-267, 294-316,321-343, 354-378, 382-452, 477-488, 529-536, 555-569, 584-591, 593-612,620-627, 632-640, 647-654, 671-680, 698-704, 723-730, 732-750, 769-775,781-788, 822-852 and 505-525 of Seq ID No 373; 3-18 of Seq ID No 374;4-14 and 12-24 of Seq ID No 375; 4-11, 22-30 and 12-25 of Seq ID No 376;5-12 and 4-18 of Seq ID No 377; 4-28 and 7-14 of Seq ID No 378; 6-16 and8-16 of Seq ID No 379; 4-15, 18-33 and 24-36 of Seq ID No 380; 4-10,16-21 and 20-31 of Seq ID No 381; 6-19 of Seq ID No 382; 11-18 and 3-10of Seq ID No 383; 13-24 and 3-15 of Seq ID No 384; 15-27 and 7-16 of SeqID No 385; 11-16 and 1-15 of Seq ID No 386; 4-16 and 9-21 of Seq ID No387; 4-24, 40-48, 54-67 and 22-39 of Seq ID No 388; 6-30, 34-55, 62-68,78-106 and 68-74 of Seq ID No 389; 3-14 of Seq ID No 390; 9-19 and 6-21of Seq ID No 391; 4-17 and 1-9 of Seq ID No 392; 5-30 and 1-8 of Seq IDNo 393; 4-16, 23-46, 51-56 and 45-55 of Seq ID No 394; 7-16 of Seq ID No395; 2-14 of Seq ID No 396; 4-36, 43-65 and 50-62 of Seq ID No 397;10-30 and 14-21 of Seq ID No 398; 9-17 and 1-10 of Seq ID No 399; 4-12and 3-16 of Seq ID No 400; 4-15 and 5-23 of Seq ID No 401; 10-21 of SeqID No 402; 6-16 of Seq ID No 403; 4-29, 31-38 and 2-14 of Seq ID No 404;4-35 and 33-42 of Seq ID No 405; 2-17 of Seq ID No 406; 9-18, 30-35 and15-33 of Seq ID No 407; 4-9 and 6-12 of Seq ID No 408; 3-17 of Seq ID No409; 12-21, 37-44, 52-61, 72-80 and 38-48 of Seq ID No 410; 4-10, 29-44,54-61, 69-78 and 13-27 of Seq ID No 411; 13-23, 36-53 and 2-15 of Seq IDNo 412; 4-25, 28-46, 56-72, 81-99, 120-132, 134-142, 154-160 and 129-141of Seq ID No 413; 4-15, 24-33, 35-41, 64-86 and 21-33 of Seq ID No 414;9-15 and 4-13 of Seq ID No 415; 4-11, 13-19, 34-48 and 15-32 of Seq IDNo 416; 4-21 and 11-31 of Seq ID No 417; 23-57 and 38-50 of Seq ID No418; 4-32 and 3-13 of Seq ID No 419; 4-10, 13-25, 32-42, 56-68, 72-84and 26-38 of Seq ID No 420; 4-20, 31-48, 52-58, 65-71, 80-93, 99-108,114-123 and 37-49 of Seq ID No 421; 6-12, 14-20 and 3-25 of Seq ID No422; 14-25, 27-38 and 5-14 of Seq ID No 423; 4-41, 57-105, 109-118,123-136, 144-152 and 86-99 of Seq ID No 424; 6-19 of Seq ID No 425; 2-19of Seq ID No 426; 14-47 and 1-14 of Seq ID No 427; 4-21, 29-44 and 2-18of Seq ID No 428; 23-29 and 10-28 of Seq ID No 429; 6-16, 22-36 and11-22 of Seq ID No 430; 4-19, 30-44 and 18-27 of Seq ID No 431; 5-15,37-45, 58-65 and 38-47 of Seq ID No 432; 4-15, 23-34 and 4-15 of Seq IDNo 433; 30-36, 44-54, 79-85, 101-114, 138-152, 154-164, 170-175,179-200, 213-220, 223-240, 243-255, 258-264, 268-284 and 10-28 of Seq IDNo 434; the peptides comprising amino acid sequences of column“Identical region” of the Table 1B, especially peptides comprising aminoacid 210-226 and 738-753 of Seq ID No 449; 326-344, 326-348, 338-354,371-392, 801-809 and 877-901 of Seq ID No 450; 893-906 of Seq ID No 451;51-69 of Seq ID No 452; 110-125 of Seq ID No 453; 291-305 of Seq ID No454; 210-226 and 738-753 of Seq ID No 455; 326-344, 326-348, 338-354,371-392, 801-809 and 877-901 of Seq ID No 456; 893-906 of Seq ID No 457;51-69 of Seq ID No 458; 110-125 of Seq ID No 459; 291-305 of Seq ID No460; 32-44 of Seq ID No 461; 399-410 of Seq ID No 462; the serumreactive epitopes as specified in the column of “aa from” to “aa to” ofTable 2, especially peptides comprising amino acid 120-143, 138-161 and156-179 of Seq ID No 218; 110-129 and 168-184 of Seq ID No 219; 74-90 ofSeq ID No 222; 759-773 of Seq ID No 223; 237-260 of Seq ID No 224;265-284 of Seq ID No 225; 65-74 of Seq ID No 226; 41-50 of Seq ID No227; 163-174 of Seq ID No 229; 26-37 of Seq ID No 230; 174-189 of Seq IDNo 232; 240-256 of Seq ID No 234; 285-297 of Seq ID No 236; 238-247 ofSeq ID No 238; 491-519 of Seq ID No 239; 114-140 of Seq ID No 243;267-284 of Seq ID No 250; 439-453 of Seq ID No 252; 162-178 of Seq ID No253; 347-364 of Seq ID No 254; 699-715 of Seq ID No 255; 60-71 of Seq IDNo 256; 244-257 of Seq ID No 257; 44-63 and 57-76 of Seq ID No 258;185-196 of Seq ID No 260; 119-129 of Seq ID No 263; 182-195 of Seq ID No266; 32-44 and 424-442 of Seq ID No 267; 247-256 of Seq ID No 268;678-694, 785-805, 55-77 and 72-94 of Seq ID No 269; 210-226 of Seq ID No281; 37-59 of Seq ID No 289; 13-29 of Seq ID No 296; 136-159 of Seq IDNo 348; 205-222 of Seq ID No 349; 99-110 of Seq ID No 350; 160-176 ofSeq ID No 351; 457-470 of Seq ID No 355; 221-237 of Seq ID No 356;167-190 of Seq ID No 357; 96-120 of Seq ID No 361; 399-417, 503-519 and544-563 of Seq ID No 364; 46-68, 159-183 and 184-198 of Seq ID No 371;463-481 of Seq ID No 372; the immunogenic epitopes as specified in thecolumn of “aa from” to “aa to” of Table 4; especially peptidescomprising amino acid 110-129 and 168-184 of Seq ID No 219; 877-901,333-354, 326-344 and 801-809 of Seq ID No 277; 1-54 of Seq ID No 347;544-563, 31-51, 107-119, 399-417 and 503-519 of Seq ID No 364; 120-198of Seq ID No 218; 20-35 of Seq ID No 219; 118-201 of Seq ID No 221;48-132 of Seq ID No 242; 118-136 of Seq ID No 249; 162-178 of Seq ID No253; 347-364 of Seq ID No 254; 699-715 of Seq ID No 255; 50-76 of Seq IDNo 258; 785-819 and 44-128 of Seq ID No 269; 90-128 of Seq ID No 274;314-384 of Seq ID No 289; 327-349 of Seq ID No 293; 242-314, 405-478 and23-100 of Seq ID No 304; 129-210 of Seq ID No 305; 162-188 of Seq ID No307; 750-772 of Seq ID No 310; 1-56 of Seq ID No 335; 322-337 of Seq IDNo 337; 72-90 of Seq ID No 339; 374-395 of Seq ID No 345; 136-159 of SeqID No 348; 141-164 of Seq ID No 358; 96-157 of Seq ID No 361; 1-82 ofSeq ID No 363; 489-556 of Seq ID No 364; 159-183 and 49-133 of Seq ID No371; The peptides comprising amino acid sequences of column “predictedimmunogenic aa” and “location of identified immunogenic region (aa)” ofTable 5, especially peptides comprising amino acid 4-26, 35-41, 53-61,73-84, 103-108, 114-120, 140-146, 156-162, 192-208, 214-219, 227-233,239-252, 260-268, 284-297, 1-48 and 113-133 of Seq ID No 475; 4-27,38-44, 50-56, 59-64, 72-79, 83-89, 92-97, 108-116, 123-148, 152-167,183-196, 200-220, 232-244, 255-261, 265-274, 282-302, 309-317, 1-79 and231-302 of Seq ID No 476; 6-28, 66-72, 85-105, 115-121, 144-151,160-170, 176-185, 223-230, 252-288, 296-310, 319-333, 367-374, 458-464,471-480, 483-488, 520-528, 530-549, 559-564, 593-601, 606-616, 636-643,655-662, 676-682, 684-699, 719-726, 735-750, 757-764, 777-785, 799-810,812-843, 846-853, 868-873, 880-889, 891-899, 909-929, 934-940, 963-969,998-1004, 1007-1014, 1016-1022, 1030-1046, 1-80 and 808-821 of Seq ID No477; 7-24, 35-41, 75-81, 91-114, 122-132, 137-144, 148-156, 183-192,194-200, 212-228, 233-238, 251-258, 275-295, 326-332, 337-346, 1-79 and305-321 of Seq ID No 478; 31-38, 42-52, 66-72, 86-92, 98-104, 115-122,127-146, 154-164, 169-187, 198-212, 225-237, 255-269, 13-92 and 135-142of Seq ID No 479; 4-36, 39-49, 63-69, 71-77, 81-88, 123-131, 133-139,160-169, 174-180, 188-194, 210-217, 273-278, 289-300, 317-334, 336-341,383-401, 425-438, 1-68, 212-270 and 402-446 of Seq ID No 480; 21-29,31-42, 49-63, 72-79, 81-93, 112-132, 159-165, 188-195, 197-232, 262-267,279-286, 294-301, 318-326, 348-366, 381-405, 409-426, 436-465, 471-480,484-492, 497-505, 521-544, 554-561, 567-577, 581-589, 601-609, 611-622,636-651, 653-667, 669-685, 700-708, 716-722, 729-744, 749-766, 780-786,789-811, 814-864, 1-57 and 84-106 of Seq ID No 481; 6-24, 35-48, 57-63,72-78, 87-92, 113-119, 123-137, 147-153, 173-181, 212-233 and 1-124 ofSeq ID No 482; 13-34, 62-69, 78-83, 86-91, 98-104, 107-115, 146-159,179-188, 195-205, 209-221, 226-233, 239-253, 276-282, 284-294, 297-308,331-354, 375-382, 388-399, 421-433, 449-458, 464-469, 472-491, 508-513,525-531, 534-550, 575-593, 601-618, 629-635, 654-661, 666-680, 706-721,723-740, 771-805, 810-830, 845-851 and 1-84 of Seq ID No 483; 4-32,45-64, 73-83, 86-92, 100-111, 125-147, 157-163, 170-175, 177-188,226-232, 245-252, 258-274, 320-335, 348-359 and 1-71 of Seq ID No 484;13-40, 43-71, 76-83, 87-101, 109-119, 125-156, 162-175, 182-219,226-232, 240-262, 270-287, 306-318, 326-342, 344-408, 414-444, 449-456and 1-51 of Seq ID No 485; 4-16, 18-34, 45-54, 99-108, 134-140, 203-212,241-257, 266-274, 279-291, 308-315, 330-336, 355-370, 374-382, 402-410,428-455, 466-472, 474-480, 531-554, 560-566, 572-580, 597-618, 632-660,664-674, 676-685, 691-705, 708-735, 750-768, 1-87 and 342-480 of Seq IDNo 486; The serum reactive epitopes as specified in the column of “aafrom” to “aa to” of Table 6, especially peptides comprising amino acid115-132 and 1-26 of Seq ID No 475; 33-55 of Seq ID No 476; 1-25 of SeqID No 478; 37-61 of Seq ID No 479; 1-24 of Seq ID No 480; 1-23 of Seq IDNo 481; 46-60 of Seq ID No 482; 1-28, 23-50 and 45-71 of Seq ID No 483;1-22 and 17-38 of Seq ID No 484; 1-22 and 17-38 of Seq ID No 485; 1-27,22-47 and 422-447 of Seq ID No 486; The immunogenic epitopes asspecified in the column of “aa from” to “aa to” of Table 7, especiallypeptides comprising amino acid 115-132 and 1-47 of Seq ID No 475; 1-55of Seq ID No 476; 22-85 of Seq ID No 477; 307-320 and 1-44 of Seq ID No478; 15-76 and 40-92 of Seq ID No 479; 1-59, 213-269 and 403-445 of SeqID No 480; 1-56 and 85-105 of Seq ID No 481; 37-121 of Seq ID No 482;1-71 of Seq ID No 483; 1-38 of Seq ID No 484; 1-38 of Seq ID No 485;1-47 of Seq ID No 486 and fragments comprising at least 6, preferablymore than 8, especially more than 10 aa and preferably not more than 70,50, 40, 20, 15 or 11 aa of said sequences. All these fragmentsindividually and each independently form a preferred selected aspect ofthe present invention.

All linear hyperimmune serum reactive fragments of a particular antigenmay be identified by analysing the entire sequence of the proteinantigen by a set of peptides overlapping by 1 amino acid with a lengthof at least 10 amino acids. Subsequently, non-linear epitopes can beidentified by analysis of the protein antigen with hyperimmune serausing the expressed full-length protein or domain polypeptides thereof.Assuming that a distinct domain of a protein is sufficient to form the3D structure independent from the native protein, the analysis of therespective recombinant or synthetically produced domain polypeptide withhyperimmune serum would allow the identification of conformationalepitopes within the individual domains of multi-domain proteins. Forthose antigens where a domain possesses linear as well as conformationalepitopes, competition experiments with peptides corresponding to thelinear epitopes may be used to confirm the presence of conformationalepitopes.

It will be appreciated that the invention also relates to, among others,nucleic acid molecules encoding the aforementioned fragments, nucleicacid molecules that hybridize to nucleic acid molecules encoding thefragments, particularly those that hybridize under stringent conditions,and nucleic acid molecules, such as PCR primers, for amplifying nucleicacid molecules that encode the fragments. In these regards, preferrednucleic acid molecules are those that correspond to the preferredfragments, as discussed above.

The present invention also relates to vectors, which comprise a nucleicacid molecule or nucleic acid molecules of the present invention, hostcells which are genetically engineered with vectors of the invention andthe production of hyperimmune serum reactive antigens and fragmentsthereof by recombinant techniques.

A great variety of expression vectors can be used to express ahyperimmune serum reactive antigen or fragment thereof according to thepresent invention. Generally, any vector suitable to maintain, propagateor express nucleic acids to express a polypeptide in a host may be usedfor expression in this regard. In accordance with this aspect of theinvention the vector may be, for example, a plasmid vector, a single ordouble-stranded phage vector, a single or double-stranded RNA or DNAviral vector. Starting plasmids disclosed herein are either commerciallyavailable, publicly available, or can be constructed from availableplasmids by routine application of well-known, published procedures.Preferred among vectors, in certain respects, are those for expressionof nucleic acid molecules and hyperimmune serum reactive antigens orfragments thereof of the present invention. Nucleic acid constructs inhost cells can be used in a conventional manner to produce the geneproduct encoded by the recombinant sequence. Alternatively, thehyperimmune serum reactive antigens and fragments thereof of theinvention can be synthetically produced by conventional peptidesynthesizers. Mature proteins can be expressed in mammalian cells,yeast, bacteria, or other cells under the control of appropriatepromoters. Cell-free translation systems can also be employed to producesuch proteins using RNAs derived from the DNA construct of the presentinvention.

Host cells can be genetically engineered to incorporate nucleic acidmolecules and express nucleic acid molecules of the present invention.Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtillis cells; fungal cells, such as yeast cells and Aspergilluscells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, Hela, C127, 3T3, BHK, 293 and Bowesmelanoma cells; and plant cells.

The invention also provides a process for producing a S. agalactiaehyperimmune serum reactive antigen and a fragment thereof comprisingexpressing from the host cell a hyperimmune serum reactive antigen orfragment thereof encoded by the nucleic acid molecules provided by thepresent invention. The invention further provides a process forproducing a cell, which expresses a S. agalactiae hyperimmune serumreactive antigen or a fragment thereof comprising transforming ortransfecting a suitable host cell with the vector according to thepresent invention such that the transformed or transfected cellexpresses the polypeptide encoded by the nucleic acid contained in thevector.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N- or C-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, regions may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stability orto facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilize orpurify polypeptides. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobin molecules together with another proteinor part thereof. In drug discovery, for example, proteins have beenfused with antibody Fc portions for the purpose of high-throughoutscreening assays to identify antagonists. See for example, {Bennett, D.et al., 1995} and {Johanson, K. et al., 1995}.

The S. agalactiae hyperimmune serum reactive antigen or a fragmentthereof can be recovered and purified from recombinant cell cultures bywell-known methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography and lectin chromatography.

The hyperimmune serum reactive antigens and fragments thereof accordingto the present invention can be produced by chemical synthesis as wellas by biotechnological means. The latter comprise the transfection ortransformation of a host cell with a vector containing a nucleic acidaccording to the present invention and the cultivation of thetransfected or transformed host cell under conditions, which are knownto the ones skilled in the art. The production method may also comprisea purification step in order to purify or isolate the polypeptide to bemanufactured. In a preferred embodiment the vector is a vector accordingto the present invention.

The hyperimmune serum reactive antigens and fragments thereof accordingto the present invention may be used for the detection of the organismor organisms in a sample containing these organisms or polypeptidesderived thereof. Preferably such detection is for diagnosis, morepreferable for the diagnosis of a disease, most preferably for thediagnosis of a diseases related or linked to the presence or abundanceof Gram-positive bacteria, especially bacteria selected from the groupcomprising streptococci, staphylococci and lactococci. More preferably,the microorganisms are selected from the group comprising Streptococcuspneumoniae, Streptococcus pyogenes and Streptococcus mutans, especiallythe microorganism is Streptococcus pyogenes.

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of thehyperimmune serum reactive antigens and fragments thereof of the presentinvention in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression of the polypeptidecompared to normal control tissue samples may be used to detect thepresence of an infection, for example, and to identify the infectingorganism. Assay techniques that can be used to determine levels of apolypeptide, in a sample derived from a host are well known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Among these, ELISAs frequently are preferred. An ELISA assay initiallycomprises preparing an antibody specific to the polypeptide, preferablya monoclonal antibody. In addition, a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached to a detectable reagent such as radioactive, fluorescent orenzymatic reagent, such as horseradish peroxidase enzyme.

The hyperimmune serum reactive antigens and fragments thereof accordingto the present invention may also be used for the purpose of or inconnection with an array. More particularly, at least one of thehyperimmune serum reactive antigens and fragments thereof according tothe present invention may be immobilized on a support. Said supporttypically comprises a variety of hyperimmune serum reactive antigens andfragments thereof whereby the variety may be created by using one orseveral of the hyperimmune serum reactive antigens and fragments thereofaccording to the present invention and/or hyperimmune serum reactiveantigens and fragments thereof being different. The characterizingfeature of such array as well as of any array in general is the factthat at a distinct or predefined region or position on said support or asurface thereof, a distinct polypeptide is immobilized. Because of thisany activity at a distinct position or region of an array can becorrelated with a specific polypeptide. The number of differenthyperimmune serum reactive antigens and fragments thereof immobilized ona support may range from as little as 10 to several 1000 differenthyperimmune serum reactive antigens and fragments thereof. The densityof hyperimmune serum reactive antigens and fragments thereof per cm² isin a preferred embodiment as little as 10 peptides/polypeptides per cm²to at least 400 different peptides/polypeptides per cm² and moreparticularly at least 1000 different hyperimmune serum reactive antigensand fragments thereof per cm².

The manufacture of such arrays is known to the one skilled in the artand, for example, described in U.S. Pat. No. 5,744,309. The arraypreferably comprises a planar, porous or non-porous solid support havingat least a first surface. The hyperimmune serum reactive antigens andfragments thereof as disclosed herein, are immobilized on said surface.Preferred support materials are, among others, glass or cellulose. It isalso within the present invention that the array is used for any of thediagnostic applications described herein. Apart from the hyperimmuneserum reactive antigens and fragments thereof according to the presentinvention also the nucleic acid molecules according to the presentinvention may be used for the generation of an array as described above.This applies as well to an array made of antibodies, preferablymonoclonal antibodies as, among others, described herein.

In a further aspect the present invention relates to an antibodydirected to any of the hyperimmune serum reactive antigens and fragmentsthereof, derivatives or fragments thereof according to the presentinvention. The present invention includes, for example, monoclonal andpolyclonal antibodies, chimeric, single chain, and humanized antibodies,as well as Fab fragments, or the product of a Fab expression library. Itis within the present invention that the antibody may be chimeric, i. e.that different parts thereof stem from different species or at least therespective sequences are taken from different species.

Antibodies generated against the hyperimmune serum reactive antigens andfragments thereof corresponding to a sequence of the present inventioncan be obtained by direct injection of the hyperimmune serum reactiveantigens and fragments thereof into an animal or by administering thehyperimmune serum reactive antigens and fragments thereof to an animal,preferably a non-human. The antibody so obtained will then bind thehyperimmune serum reactive antigens and fragments thereof itself. Inthis manner, even a sequence encoding only a fragment of a hyperimmuneserum reactive antigen and fragments thereof can be used to generateantibodies binding the whole native hyperimmune serum reactive antigenand fragments thereof. Such antibodies can then be used to isolate thehyperimmune serum reactive antigens and fragments thereof from tissueexpressing those hyperimmune serum reactive antigens and fragmentsthereof.

For preparation of monoclonal antibodies, any technique known in theart, which provides antibodies produced by continuous cell line culturescan be used (as described originally in {Kohler, G. et al., 1975}.

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic hyperimmune serum reactive antigens and fragments thereofaccording to this invention. Also, transgenic mice, or other organismssuch as other mammals, may be used to express humanized antibodies toimmunogenic hyperimmune serum reactive antigens and fragments thereofaccording to this invention.

Alternatively, phage display technology or ribosomal display could beutilized to select antibody genes with binding activities towards thehyperimmune serum reactive antigens and fragments thereof either fromrepertoires of PCR amplified v-genes of lymphocytes from humans screenedfor possessing respective target antigens or from naïve libraries{McCafferty, J. et al., 1990}; {Marks, J. et al., 1992}. The affinity ofthese antibodies can also be improved by chain shuffling {Clackson, T.et al., 1991}.

If two antigen binding domains are present, each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the hyperimmune serum reactive antigens and fragmentsthereof or purify the hyperimmune serum reactive antigens and fragmentsthereof of the present invention by attachment of the antibody to asolid support for isolation and/or purification by affinitychromatography.

Thus, among others, antibodies against the hyperimmune serum reactiveantigens and fragments thereof of the present invention may be employedto inhibit and/or treat infections, particularly bacterial infectionsand especially infections arising from S. agalactiae.

Hyperimmune serum reactive antigens and fragments thereof includeantigenically, epitopically or immunologically equivalent derivatives,which form a particular aspect of this invention. The term“antigenically equivalent derivative” as used herein encompasses ahyperimmune serum reactive antigen and fragments thereof or itsequivalent which will be specifically recognized by certain antibodieswhich, when raised to the protein or hyperimmune serum reactive antigenand fragments thereof according to the present invention, interfere withthe interaction between pathogen and mammalian host. The term“immunologically equivalent derivative” as used herein encompasses apeptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe interaction between pathogen and mammalian host.

The hyperimmune serum reactive antigens and fragments thereof, such asan antigenically or immunologically equivalent derivative or a fusionprotein thereof can be used as an antigen to immunize a mouse or otheranimal such as a rat or chicken. The fusion protein may providestability to the hyperimmune serum reactive antigens and fragmentsthereof. The antigen may be associated, for example by conjugation, withan immunogenic carrier protein, for example bovine serum albumin (BSA)or keyhole limpet haemocyanin (KLH). Alternatively, an antigenic peptidecomprising multiple copies of the protein or hyperimmune serum reactiveantigen and fragments thereof, or an antigenically or immunologicallyequivalent hyperimmune serum reactive antigen and fragments thereof, maybe sufficiently antigenic to improve immunogenicity so as to obviate theuse of a carrier.

Preferably the antibody or derivative thereof is modified to make itless immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized”, wherein thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in {Jones, P. et al., 1986} or {Tempest, P. et al., 1991}.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscle, delivery of DNA complexed withspecific protein carriers, coprecipitation of DNA with calciumphosphate, encapsulation of DNA in various forms of liposomes, particlebombardment {Tang, D. et al., 1992}, {Eisenbraun, M. et al., 1993} andin vivo infection using cloned retroviral vectors {Seeger, C. et al.,1984}.

In a further aspect the present invention relates to a peptide bindingto any of the hyperimmune serum reactive antigens and fragments thereofaccording to the present invention, and a method for the manufacture ofsuch peptides whereby the method is characterized by the use of thehyperimmune serum reactive antigens and fragments thereof according tothe present invention and the basic steps are known to the one skilledin the art.

Such peptides may be generated by using methods according to the stateof the art such as phage display or ribosome display. In case of phagedisplay, basically a library of peptides is generated, in form ofphages, and this kind of library is contacted with the target molecule,in the present case a hyperimmune serum reactive antigen and fragmentsthereof according to the present invention. Those peptides binding tothe target molecule are subsequently removed, preferably as a complexwith the target molecule, from the respective reaction. It is known tothe one skilled in the art that the binding characteristics, at least toa certain extent, depend on the particularly realized experimentalset-up such as the salt concentration and the like. After separatingthose peptides binding to the target molecule with a higher affinity ora bigger force, from the non-binding members of the library, andoptionally also after removal of the target molecule from the complex oftarget molecule and peptide, the respective peptide(s) may subsequentlybe characterised. Prior to the characterisation optionally anamplification step is realized such as, e. g. by propagating the peptideencoding phages. The characterisation preferably comprises thesequencing of the target binding peptides. Basically, the peptides arenot limited in their lengths, however, preferably peptides having alengths from about 8 to 20 amino acids are preferably obtained in therespective methods. The size of the libraries may be about 10² to 10¹⁸,preferably 10⁸ to 10¹⁵ different peptides, however, is not limitedthereto.

A particular form of target binding hyperimmune serum reactive antigensand fragments thereof are the so-called “anticalines” which are, amongothers, described in German patent application DE 197 42 706.

In a further aspect the present invention relates to functional nucleicacids interacting with any of the hyperimmune serum reactive antigensand fragments thereof according to the present invention, and a methodfor the manufacture of such functional nucleic acids whereby the methodis characterized by the use of the hyperimmune serum reactive antigensand fragments thereof according to the present invention and the basicsteps are known to the one skilled in the art. The functional nucleicacids are preferably aptamers and spiegelmers.

Aptamers are D-nucleic acids, which are either single stranded or doublestranded and which specifically interact with a target molecule. Themanufacture or selection of aptamers is, e.g. described in Europeanpatent EP 0 533 838. Basically the following steps are realized. First,a mixture of nucleic acids, i. e. potential aptamers, is providedwhereby each nucleic acid typically comprises a segment of several,preferably at least eight subsequent randomised nucleotides. Thismixture is subsequently contacted with the target molecule whereby thenucleic acid(s) bind to the target molecule, such as based on anincreased affinity towards the target or with a bigger force thereto,compared to the candidate mixture. The binding nucleic acid(s) are/issubsequently separated from the remainder of the mixture. Optionally,the thus obtained nucleic acid(s) is amplified using, e.g. polymerasechain reaction. These steps may be repeated several times giving at theend a mixture having an increased ratio of nucleic acids specificallybinding to the target from which the final binding nucleic acid is thenoptionally selected. These specifically binding nucleic acid(s) arereferred to as aptamers. It is obvious that at any stage of the methodfor the generation or identification of the aptamers samples of themixture of individual nucleic acids may be taken to determine thesequence thereof using standard techniques. It is within the presentinvention that the aptamers may be stabilized such as, e. g., byintroducing defined chemical groups which are known to the one skilledin the art of generating aptamers. Such modification may for examplereside in the introduction of an amino group at the 2′-position of thesugar moiety of the nucleotides. Aptamers are currently used astherapeutical agents. However, it is also within the present inventionthat the thus selected or generated aptamers may be used for targetvalidation and/or as lead substance for the development of medicaments,preferably of medicaments based on small molecules. This is actuallydone by a competition assay whereby the specific interaction between thetarget molecule and the aptamer is inhibited by a candidate drug wherebyupon replacement of the aptamer from the complex of target and aptamerit may be assumed that the respective drug candidate allows a specificinhibition of the interaction between target and aptamer, and if theinteraction is specific, said candidate drug will, at least inprinciple, be suitable to block the target and thus decrease itsbiological availability or activity in a respective system comprisingsuch target. The thus obtained small molecule may then be subject tofurther derivatisation and modification to optimise its physical,chemical, biological and/or medical characteristics such as toxicity,specificity, biodegradability and bioavailability.

Spiegelmers and their generation or manufacture is based on a similarprinciple. The manufacture of spiegelmers is described in internationalpatent application WO 98/08856. Spiegelmers are L-nucleic acids, whichmeans that they are composed of L-nucleotides rather than D-nucleotidesas aptamers are. Spiegelmers are characterized by the fact that theyhave a very high stability in biological systems and, comparable toaptamers, specifically interact with the target molecule against whichthey are directed. In the process of generating spiegelmers, aheterogenous population of D-nucleic acids is created and thispopulation is contacted with the optical antipode of the targetmolecule, in the present case for example with the D-enantiomer of thenaturally occurring L-enantiomer of the hyperimmune serum reactiveantigens and fragments thereof according to the present invention.Subsequently, those D-nucleic acids are separated which do not interactwith the optical antipode of the target molecule. But those D-nucleicacids interacting with the optical antipode of the target molecule areseparated, optionally identified and/or sequenced and subsequently thecorresponding L-nucleic acids are synthesized based on the nucleic acidsequence information obtained from the D-nucleic acids. These L-nucleicacids which are identical in terms of sequence with the aforementionedD-nucleic acids interacting with the optical antipode of the targetmolecule, will specifically interact with the naturally occurring targetmolecule rather than with the optical antipode thereof. Similar to themethod for the generation of aptamers it is also possible to repeat thevarious steps several times and thus to enrich those nucleic acidsspecifically interacting with the optical antipode of the targetmolecule.

In a further aspect the present invention relates to functional nucleicacids interacting with any of the nucleic acid molecules according tothe present invention, and a method for the manufacture of suchfunctional nucleic acids whereby the method is characterized by the useof the nucleic acid molecules and their respective sequences accordingto the present invention and the basic steps are known to the oneskilled in the art. The functional nucleic acids are preferablyribozymes, antisense oligonucleotides and siRNA.

Ribozymes are catalytically active nucleic acids, which preferablyconsist of RNA, which basically comprises two moieties. The first moietyshows a catalytic activity whereas the second moiety is responsible forthe specific interaction with the target nucleic acid, in the presentcase the nucleic acid coding for the hyperimmune serum reactive antigensand fragments thereof according to the present invention. Uponinteraction between the target nucleic acid and the second moiety of theribozyme, typically by hybridisation and Watson-Crick base pairing ofessentially complementary stretches of bases on the two hybridisingstrands, the catalytically active moiety may become active which meansthat it catalyses, either intramolecularly or intermolecularly, thetarget nucleic acid in case the catalytic activity of the ribozyme is aphosphodiesterase activity. Subsequently, there may be a furtherdegradation of the target nucleic acid, which in the end results in thedegradation of the target nucleic acid as well as the protein derivedfrom the said target nucleic acid. Ribozymes, their use and designprinciples are known to the one skilled in the art, and, for exampledescribed in {Doherty, E. et al., 2001} and {Lewin, A. et al., 2001}.

The activity and design of antisense oligonucleotides for themanufacture of a medicament and as a diagnostic agent, respectively, isbased on a similar mode of action. Basically, antisense oligonucleotideshybridize based on base complementarity, with a target RNA, preferablywith a mRNA, thereby activating RNase H. RNase H is activated by bothphosphodiester and phosphorothioate-coupled DNA. Phosphodiester-coupledDNA, however, is rapidly degraded by cellular nucleases with theexception of phosphorothioate-coupled DNA. These resistant,non-naturally occurring DNA derivatives do not inhibit RNase H uponhybridisation with RNA. In other words, antisense polynucleotides areonly effective as DNA RNA hybride complexes. Examples for this kind ofantisense oligonucleotides are described, among others, in U.S. Pat. No.5,849,902 and U.S. Pat. No. 5,989,912. In other words, based on thenucleic acid sequence of the target molecule which in the present caseare the nucleic acid molecules for the hyperimmune serum reactiveantigens and fragments thereof according to the present invention,either from the target protein from which a respective nucleic acidsequence may in principle be deduced, or by knowing the nucleic acidsequence as such, particularly the mRNA, suitable antisenseoligonucleotides may be designed base on the principle of basecomplementarity.

Particularly preferred are antisense-oligonucleotides, which have ashort stretch of phosphorothioate DNA (3 to 9 bases). A minimum of 3 DNAbases is required for activation of bacterial RNase H and a minimum of 5bases is required for mammalian RNase H activation. In these chimericoligonucleotides there is a central region that forms a substrate forRNase H that is flanked by hybridising “arms” comprised of modifiednucleotides that do not form substrates for RNase H. The hybridisingarms of the chimeric oligonucleotides may be modified such as by2′-O-methyl or 2′-fluoro. Alternative approaches used methylphosphonateor phosphoramidate linkages in said arms. Further embodiments of theantisense oligonucleotide useful in the practice of the presentinvention are P-methoxyoligonucleotides, partialP-methoxyoligodeoxyribonucleotides or P-methoxyoligonucleotides.

Of particular relevance and usefulness for the present invention arethose antisense oligonucleotides as more particularly described in theabove two mentioned U.S. patents. These oligonucleotides contain nonaturally occurring 5′→3′-linked nucleotides. Rather theoligonucleotides have two types of nucleotides:2′-deoxyphosphorothioate, which activate RNase H, and 2′-modifiednucleotides, which do not. The linkages between the 2′-modifiednucleotides can be phosphodiesters, phosphorothioate orP-ethoxyphosphodiester. Activation of RNase H is accomplished by acontiguous RNase H-activating region, which contains between 3 and 52′-deoxyphosphorothioate nucleotides to activate bacterial RNase H andbetween 5 and 10 2′- deoxyphosphorothioate nucleotides to activateeucaryotic and, particularly, mammalian RNase H. Protection fromdegradation is accomplished by making the 5′ and 3′ terminal baseshighly nuclease resistant and, optionally, by placing a 3′ terminalblocking group.

More particularly, the antisense oligonucleotide comprises a 5′ terminusand a 3′ terminus; and from position 11 to 59 5′→3′-linked nucleotidesindependently selected from the group consisting of 2′-modifiedphosphodiester nucleotides and 2′-modified P-alkyloxyphosphotriesternucleotides; and wherein the 5′-terminal nucleoside is attached to anRNase H-activating region of between three and ten contiguousphosphorothioate-linked deoxyribonucleotides, and wherein the3′-terminus of said oligonucleotide is selected from the groupconsisting of an inverted deoxyribonucleotide, a contiguous stretch ofone to three phosphorothioate 2′-modified ribonucleotides, a biotingroup and a P-alkyloxyphosphotriester nucleotide.

Also an antisense oligonucleotide may be used wherein not the 5′terminal nucleoside is attached to an RNase H-activating region but the3′ terminal nucleoside as specified above. Also, the 5′ terminus isselected from the particular group rather than the 3′ terminus of saidoligonucleotide.

The nucleic acids as well as the hyperimmune serum reactive antigens andfragments thereof according to the present invention may be used as orfor the manufacture of pharmaceutical compositions, especially vaccines.Preferably such pharmaceutical composition, preferably vaccine is forthe prevention or treatment of diseases caused by, related to orassociated with S. agalactiae. In so far another aspect of the inventionrelates to a method for inducing an immunological response in anindividual, particularly a mammal, which comprises inoculating theindividual with the hyperimmune serum reactive antigens and fragmentsthereof of the invention, or a fragment or variant thereof, adequate toproduce antibodies to protect said individual from infection,particularly streptococcal infection and most particularly S. agalactiaeinfections.

Yet another aspect of the invention relates to a method of inducing animmunological response in an individual which comprises, through genetherapy or otherwise, delivering a nucleic acid functionally encodinghyperimmune serum reactive antigens and fragments thereof, or a fragmentor a variant thereof, for expressing the hyperimmune serum reactiveantigens and fragments thereof, or a fragment or a variant thereof invivo in order to induce an immunological response to produce antibodiesor a cell mediated T cell response, either cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether thatdisease is already established within the individual or not. One-way ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable of having inducedwithin it an immunological response, induces an immunological responsein such host, wherein the composition comprises recombinant DNA whichcodes for and expresses an antigen of the hyperimmune serum reactiveantigens and fragments thereof of the present invention. Theimmunological response may be used therapeutically or prophylacticallyand may take the form of antibody immunity or cellular immunity such asthat arising from CTL or CD4+T cells.

The hyperimmune serum reactive antigens and fragments thereof of theinvention or a fragment thereof may be fused with a co-protein which maynot by itself produce antibodies, but is capable of stabilizing thefirst protein and producing a fused protein which will have immunogenicand protective properties. This fused recombinant protein preferablyfurther comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilise the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

Also, provided by this invention are methods using the described nucleicacid molecule or particular fragments thereof in such geneticimmunization experiments in animal models of infection with S.agalactiae. Such fragments will be particularly useful for identifyingprotein epitopes able to provoke a prophylactic or therapeutic immuneresponse. This approach can allow for the subsequent preparation ofmonoclonal antibodies of particular value from the requisite organ ofthe animal successfully resisting or clearing infection for thedevelopment of prophylactic agents or therapeutic treatments of S.agalactiae infection in mammals, particularly humans.

The hyperimmune serum reactive antigens and fragments thereof may beused as an antigen for vaccination of a host to produce specificantibodies which protect against invasion of bacteria, for example byblocking adherence of bacteria to damaged tissue. Examples of tissuedamage include wounds in skin or connective tissue and mucosal tissuescaused e.g. by viral infection (esp. respiratory, such as the flu)mechanical, chemical or thermal damage or by implantation of indwellingdevices, or wounds in the mucous membranes, such as the mouth, mammaryglands, urethra or vagina.

The present invention also includes a vaccine formulation, whichcomprises the immunogenic recombinant protein together with a suitablecarrier. Since the protein may be broken down in the stomach, it ispreferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous,intradermal intranasal or transdermal Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with thebodily fluid, preferably the blood, of the individual; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials, and maybe stored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in-water systems and other systems known inthe art. The dosage will depend on the specific activity of the vaccineand can be readily determined by routine experimentation.

According to another aspect, the present invention relates to apharmaceutical composition comprising such a hyperimmune serum-reactiveantigen or a fragment thereof as provided in the present invention forS. agalactiae. Such a pharmaceutical composition may comprise onepreferably at least two or more hyperimmune serum reactive antigens orfragments thereof against S. agalactiae. Optionally, such S. agalactiaehyperimmune serum reactive antigens or fragments thereof may also becombined with antigens against other pathogens in a combinationpharmaceutical composition. Preferably, said pharmaceutical compositionis a vaccine for preventing or treating an infection caused by S.agalactiae and/or other pathogens against which the antigens have beenincluded in the vaccine.

According to a further aspect, the present invention relates to apharmaceutical composition comprising a nucleic acid molecule encoding ahyperimmune serum-reactive antigen or a fragment thereof as identifiedabove for S. agalactiae. Such a pharmaceutical composition may compriseone or more nucleic acid molecules encoding hyperimmune serum reactiveantigens or fragments thereof against S. agalactiae. Optionally, such S.agalactiae nucleic acid molecules encoding hyperimmune serum reactiveantigens or fragments thereof may also be combined with nucleic acidmolecules encoding antigens against other pathogens in a combinationpharmaceutical composition. Preferably, said pharmaceutical compositionis a vaccine for preventing or treating an infection caused by S.agalactiae and/or other pathogens against which the antigens have beenincluded in the vaccine.

The pharmaceutical composition may contain any suitable auxiliarysubstances, such as buffer substances, stabilisers or further activeingredients, especially ingredients known in connection ofpharmaceutical composition and/or vaccine production.

A preferable carrier/or excipient for the hyperimmune serum-reactiveantigens, fragments thereof or a coding nucleic acid molecule thereofaccording to the present invention is an immunostimulatory compound forfurther stimulating the immune response to the given hyperimmuneserum-reactive antigen, fragment thereof or a coding nucleic acidmolecule thereof. Preferably the immunostimulatory compound in thepharmaceutical preparation according to the present invention isselected from the group of polycationic substances, especiallypolycationic peptides, immunostimulatory nucleic acids molecules,preferably immunostimulatory deoxynucleotides, alum, Freund's completeadjuvants, Freund's incomplete adjuvants, neuroactive compounds,especially human growth hormone, or combinations thereof.

It is also within the scope of the present invention that thepharmaceutical composition, especially vaccine, comprises apart from thehyperimmune serum reactive antigens, fragments thereof and/or codingnucleic acid molecules thereof according to the present invention othercompounds which are biologically or pharmaceutically active. Preferably,the vaccine composition comprises at least one polycationic peptide. Thepolycationic compound(s) to be used according to the present inventionmay be any polycationic compound, which shows the characteristic effectsaccording to the WO 97/30721. Preferred polycationic compounds areselected from basic polypeptides, organic polycations, basic polyaminoacids or mixtures thereof. These polyamino acids should have a chainlength of at least 4 amino acid residues (WO 97/30721). Especiallypreferred are substances like polylysine, polyarginine and polypeptidescontaining more than 20%, especially more than 50% of basic amino acidsin a range of more than 8, especially more than 20, amino acid residuesor mixtures thereof. Other preferred polycations and theirpharmaceutical compositions are described in WO 97/30721 (e.g.polyethyleneimine) and WO 99/38528. Preferably these polypeptidescontain between 20 and 500 amino acid residues, especially between 30and 200 residues.

These polycationic compounds may be produced chemically or recombinantlyor may be derived from natural sources.

Cationic (poly)peptides may also be anti-microbial with properties asreviewed in {Ganz, T., 1999}. These (poly)peptides may be of prokaryoticor animal or plant origin or may be produced chemically or recombinantly(WO 02/13857). Peptides may also belong to the class of defensins (WO02/13857). Sequences of such peptides can be, for example, found in theAntimicrobial Sequences Database under the following internet address:

-   -   http://www.bbcm.univ.trieste.it/-tossi/pag2.html

Such host defense peptides or defensives are also a preferred form ofthe polycationic polymer according to the present invention. Generally,a compound allowing as an end product activation (or down-regulation) ofthe adaptive immune system, preferably mediated by APCs (includingdendritic cells) is used as polycationic polymer.

Especially preferred for use as polycationic substances in the presentinvention are cathelicidin derived antimicrobial peptides or derivativesthereof (International patent application WO 02/13857, incorporatedherein by reference), especially antimicrobial peptides derived frommammalian cathelicidin, preferably from human, bovine or mouse.

Polycationic compounds derived from natural sources include HIV-REV orHIV-TAT (derived cationic peptides, antennapedia peptides, chitosan orother derivatives of chitin) or other peptides derived from thesepeptides or proteins by biochemical or recombinant production. Otherpreferred polycationic compounds are cathelin or related or derivedsubstances from cathelin. For example, mouse cathelin is a peptide whichhas the amino acid sequence NH₂-RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPECOOH(SEQ ID NO:488). Related or derived cathelin substances contain thewhole or parts of the cathelin sequence with at least 15-20 amino acidresidues. Derivations may include the substitution or modification ofthe natural amino acids by amino acids which are not among the 20standard amino acids. Moreover, further cationic residues may beintroduced into such cathelin molecules. These cathelin molecules arepreferred to be combined with the antigen. These cathelin moleculessurprisingly have turned out to be also effective as an adjuvant for anantigen without the addition of further adjuvants. It is thereforepossible to use such cathelin molecules as efficient adjuvants invaccine formulations with or without further immunoactivatingsubstances.

Another preferred polycationic substance to be used according to thepresent invention is a synthetic peptide containing at least 2KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids(International patent application WO 02/32451, incorporated herein byreference).

The pharmaceutical composition of the present invention may furthercomprise immunostimulatory nucleic acid(s). Immunostimulatory nucleicacids are e. g. neutral or artificial CpG containing nucleic acids,short stretches of nucleic acids derived from non-vertebrates or in formof short oligonucleotides (ODNs) containing non-methylatedcytosine-guanine di-nucleotides (CpG) in a certain base context (e.g.described in WO 96/02555). Alternatively, also nucleic acids based oninosine and cytidine as e.g. described in the WO 01/93903, ordeoxynucleic acids containing deoxy-inosine and/or deoxyuridine residues(described in WO 01/93905 and PCT/EP 02/05448, incorporated herein byreference) may preferably be used as immunostimulatory nucleic acids forthe present invention. Preferably, the mixtures of differentimmunostimulatory nucleic acids may be used according to the presentinvention.

It is also within the present invention that any of the aforementionedpolycationic compounds is combined with any of the immunostimulatorynucleic acids as aforementioned. Preferably, such combinations areaccording to the ones as described in WO 01/93905, WO 02/32451, WO01/54720, WO 01/93903, WO 02/13857 and PCT/EP 02/05448 and the Austrianpatent application A 1924/2001, incorporated herein by reference.

In addition or alternatively such vaccine composition may comprise apartfrom the hyperimmune serum reactive antigens and fragments thereof, andthe coding nucleic acid molecules thereof according to the presentinvention a neuroactive compound. Preferably, the neuroactive compoundis human growth factor as, e.g. described in WO 01/24822. Alsopreferably, the neuroactive compound is combined with any of thepolycationic compounds and/or immunostimulatory nucleic acids asafore-mentioned.

In a further aspect the present invention is related to a pharmaceuticalcomposition. Such pharmaceutical composition is, for example, thevaccine described herein. Also a pharmaceutical composition is apharmaceutical composition which comprises any of the followingcompounds or combinations thereof: the nucleic acid molecules accordingto the present invention, the hyperimmune serum reactive antigens andfragments thereof according to the present invention, the vectoraccording to the present invention, the cells according to the presentinvention, the antibody according to the present invention, thefunctional nucleic acids according to the present invention and thebinding peptides such as the anticalines according to the presentinvention, any agonists and antagonists screened as described herein. Inconnection therewith any of these compounds may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa hyperimmune serum reactive antigen and fragments thereof of theinvention and a pharmaceutically acceptable carrier or excipient. Suchcarriers may include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, intratracheal or intradermal routes amongothers.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical application,for example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.05-5 μg antigen/per kg of body weight,and such dose is preferably administered 1-3 times and with an intervalof 1-3 weeks.

With the indicated dose range, no adverse toxicological effects shouldbe observed with the compounds of the invention, which would precludetheir administration to suitable individuals.

In a further embodiment the present invention relates to diagnostic andpharmaceutical packs and kits comprising one or more containers filledwith one or more of the ingredients of the aforementioned compositionsof the invention. The ingredient(s) can be present in a useful amount,dosage, formulation or combination. Associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, reflecting approval by the agency of the manufacture, use orsale of the product for human administration.

In connection with the present invention any disease related use asdisclosed herein such as, e. g. use of the pharmaceutical composition orvaccine, is particularly a disease or diseased condition which is causedby, linked or associated with Streptococci, more preferably, S. pyogensand pneumoniae. In connection therewith it is to be noted that S.agalactiae comprises several strains including those disclosed herein. Adisease related, caused or associated with the bacterial infection to beprevented and/or treated according to the present invention includesbesides others bacterial pharyngitis, otitis media, pneumonia,bacteremia, meningitis, peritonitis, wound infection and sepsis inhumans.

In a still further embodiment the present invention is related to ascreening method using any of the hyperimmune serum reactive antigens ornucleic acids according to the present invention. Screening methods assuch are known to the one skilled in the art and can be designed suchthat an agonist or an antagonist is screened. Preferably an antagonistis screened which in the present case inhibits or prevents the bindingof any hyperimmune serum reactive antigen and fragment thereof accordingto the present invention to an interaction partner. Such interactionpartner can be a naturally occurring interaction partner or anon-naturally occurring interaction partner.

The invention also provides a method of screening compounds to identifythose, which enhance (agonist) or block (antagonist) the function ofhyperimmune serum reactive antigens and fragments thereof or nucleicacid molecules of the present invention, such as its interaction with abinding molecule. The method of screening may involve high-throughput.

For example, to screen for agonists or antagonists, the interactionpartner of the nucleic acid molecule and nucleic acid, respectively,according to the present invention, maybe a synthetic reaction mix, acellular compartment, such as a membrane, cell envelope or cell wall, ora preparation of any thereof, may be prepared from a cell that expressesa molecule that binds to the hyperimmune serum reactive antigens andfragments thereof of the present invention. The preparation is incubatedwith labelled hyperimmune serum reactive antigens and fragments thereofin the absence or the presence of a candidate molecule, which may be anagonist or antagonist. The ability of the candidate molecule to bind thebinding molecule is reflected in decreased binding of the labelledligand. Molecules which bind gratuitously, i. e., without inducing thefunctional effects of the hyperimmune serum reactive antigens andfragments thereof, are most likely to be good antagonists. Moleculesthat bind well and elicit functional effects that are the same as orclosely related to the hyperimmune serum reactive antigens and fragmentsthereof are good agonists.

The functional effects of potential agonists and antagonists may bemeasured, for instance, by determining the activity of a reporter systemfollowing interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that of thehyperimmune serum reactive antigens and fragments thereof of the presentinvention or molecules that elicit the same effects as the hyperimmuneserum reactive antigens and fragments thereof. Reporter systems that maybe useful in this regard include but are not limited to colorimetriclabelled substrate converted into product, a reporter gene that isresponsive to changes in the functional activity of the hyperimmuneserum reactive antigens and fragments thereof, and binding assays knownin the art.

Another example of an assay for antagonists is a competitive assay thatcombines the hyperimmune serum reactive antigens and fragments thereofof the present invention and a potential antagonist with membrane-boundbinding molecules, recombinant binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. The hyperimmune serum reactiveantigens and fragments thereof can be labelled such as by radioactivityor a colorimetric compound, such that the molecule number of hyperimmuneserum reactive antigens and fragments thereof bound to a bindingmolecule or converted to product can be determined accurately to assessthe effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a hyperimmune serum reactiveantigen and fragments thereof of the invention and thereby inhibit orextinguish its activity. Potential antagonists also may be small organicmolecules, a peptide, a polypeptide such as a closely related protein orantibody that binds to the same sites on a binding molecule withoutinducing functional activity of the hyperimmune serum reactive antigensand fragments thereof of the invention.

Potential antagonists include a small molecule, which binds to andoccupies the binding site of the hyperimmune serum reactive antigens andfragments thereof thereby preventing binding to cellular bindingmolecules, such that normal biological activity is prevented. Examplesof small molecules include but are not limited to small organicmolecules, peptides or peptide-like molecules.

Other potential antagonists include antisense molecules (see {Okano, H.et al., 1991}; OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENEEXPRESSION; CRC Press, Boca Ration, Fla. (1988), for a description ofthese molecules).

Preferred potential antagonists include derivatives of the hyperimmuneserum reactive antigens and fragments thereof of the invention.

As used herein the activity of a hyperimmune serum reactive antigen andfragment thereof according to the present invention is its capability tobind to any of its interaction partner or the extent of such capabilityto bind to its or any interaction partner.

In a particular aspect, the invention provides the use of thehyperimmune serum reactive antigens and fragments thereof, nucleic acidmolecules or inhibitors of the invention to interfere with the initialphysical interaction between a pathogen and mammalian host responsiblefor sequelae of infection. In particular the molecules of the inventionmay be used: i) in the prevention of adhesion of S. agalactiae tomammalian extracellular matrix proteins at mucosal surfaces and onin-dwelling devices or to extracellular matrix proteins in wounds; ii)to block bacterial adhesion between mammalian extracellular matrixproteins and bacterial proteins which mediate tissue damage or invasioniii) or lead to evasion of immune defense; iv) to block the normalprogression of pathogenesis in infections initiated other than by theimplantation of in-dwelling devices or by other surgical techniques,e.g. through inhibiting nutrient acquisition.

Each of the DNA coding sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein upon expression can be used as a target for the screening ofantibacterial drugs. Additionally, the DNA sequences encoding the aminoterminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

The antagonists and agonists may be employed, for instance, to inhibitdiseases arising from infection with Streptococcus, especially S.agalactiae, such as sepsis.

In a still further aspect the present invention is related to anaffinity device such affinity device comprises as least a supportmaterial and any of the hyperimmune serum reactive antigens andfragments thereof according to the present invention, which is attachedto the support material. Because of the specificity of the hyperimmuneserum reactive antigens and fragments thereof according to the presentinvention for their target cells or target molecules or theirinteraction partners, the hyperimmune serum reactive antigens andfragments thereof allow a selective removal of their interactionpartner(s) from any kind of sample applied to the support materialprovided that the conditions for binding are met. The sample may be abiological or medical sample, including but not limited to, fermentationbroth, cell debris, cell preparation, tissue preparation, organpreparation, blood, urine, lymph liquid, liquor and the like.

The hyperimmune serum reactive antigens and fragments thereof may beattached to the matrix in a covalent or non-covalent manner. Suitablesupport material is known to the one skilled in the art and can beselected from the group comprising cellulose, silicon, glass, aluminium,paramagnetic beads, starch and dextrane.

The present invention is further illustrated by the following figures,examples and the sequence listing, from which further features,embodiments and advantages may be taken. It is to be understood that thepresent examples are given by way of illustration only and not by way oflimitation of the disclosure.

In connection with the present invention

FIG. 1 shows the characterization of human antibody sources for S.agalactiae.

FIG. 2 shows the characterization of the small fragment genomic library,LSAg-70, from Streptococcus agalactiae ATCC 12403.

FIG. 3 shows the selection of bacterial cells by MACS using biotinylatedhuman IgGs.

FIG. 4 shows the serotypes of the applied strains and an example for thegene distribution analysis with one of the identified antigens.

FIG. 5 shows examples for induction of epitope-specific antibodies inmice by immunization with E. coli lysates.

FIG. 6 shows examples for cell surface staining with epitope-specificantisera by flow cytometry.

FIG. 7 shows the determination of bactericidal activity of antibodiesinduced by selected epitopes in an in vitro assay.

Table 1A shows the summary of all screens performed with genomic S.agalactiae libraries and human serum. Table 1B shows antigenic proteinsidentified by sequence identity within antigenic regions of the proteinslisted in Table 1A.

Table 2 shows the summary of epitope serology analysis with human sera.

Table 3 shows the summary of the gene distribution analysis for theidentified antigens in 46 S. agalactiae strains.

Table 4 shows the summary of mouse immunogenicity experiments.

Table 5 shows the summary of all screens performed with genomic S.agalactiae libraries and human serum.

Table 6 shows the summary of epitope serology analysis with human sera.

Table 7 shows the summary of mouse immunogenicity experiments.

The figures to which it might be referred to in the specification aredescribed in the following in more details.

FIG. 1 shows the characterization of human sera and cervical secretionsfor anti-S. agalactiae antibodies as measured by immune assays. Totalanti-S. agalactiae IgG and IgA antibody levels were measured by standardELISA using total bacterial lysates or culture supernant fractionsprepared; from S. agalactiae serotype III strain ATCC 12403 as coatingantigens. (A) Results of representative experiments are shown withhealthy adult sera with total bacterial lysate proteins. Data areexpressed as ELISA units calculated from absorbance at 405 nm at a serumdilution in the linear range of detection (2.000× for IgA, 10,000 forIgG). Selected sera (out of 52) included in the healthy adultnon-pregnant serum pool (NSag8-IgG,-IgA) are indicated by bold numbers.(B) Immunoblot analysis was performed on high titer sera selected byELISA in order to ensure multiple immune reactivity with proteinantigens. Results of a representative experiment using total bacteriallysate prepared from S. agalactiae serotype III ATCC 12403 strain andselected patients' sera at 5.000× dilution are shown. Blots weredeveloped with anti-human IgG secondary antibody reagent. Low titer serawere included as negative controls. Mw: molecular weight markers. (C)shows selection of cervical secretions from noncolonized pregnant womenby immunoblot analysis. Antibodies extracted from cervical wicks werequantitated for IgA content. 2 μg IgA from each preparations were testedfor immunoreactivity using total bacterial lysate in a multi-wellblotting apparatus. Blots were developed with anti-human IgA secondaryantibodies. IgA preparation showing reactivity with GBS proteins(indicated by arrows) were selected and pooled.

FIG. 2(A) shows the fragment size distribution of the Streptococcusagalactiae ATCC 12403 small fragment genomic library, LSAg-70. Aftersequencing 576 randomly selected clones, sequences were trimmed (464) toeliminate vector residues and the numbers of clones with various genomicfragment sizes were plotted. (B) shows the graphic illustration of thedistribution of the same set of randomly sequenced clones of LSAg-70over the S. agalactiae ATCC 12403 chromosome. Rectangles indicatematching sequences to annotated ORFs and diamonds represent fullymatched clones to non-coding chromosomal sequences in +/+ or +/−orientation. Circles position all clones with chimeric sequences.Numeric distances in base pairs are indicated over the circular genomefor orientation. Partitioning of various clone sets within the libraryis given in numbers and percentage at the bottom of the figure.

FIG. 3(A) shows the MACS selection with biotinylated human IgGs. TheLSAg-70 library in pMAL9.1 was screened with 15-20 μg biotinylated IgG(PSag11-IgG, purified from human serum). As negative control, no serumwas added to the library cells for screening. Number of cells selectedafter the 1^(st), 2^(nd) and 3^(rd) elution are shown for each selectionround (upper, middle and lower panel, respectively). (B) shows thereactivity of specific clones (1-26) selected by bacterial surfacedisplay as analyzed by immunoblot analysis with the human serum IgG pool(PSag11-IgG, 4 μg/μl) used for selection by MACS at a dilution of1:3,000. As a loading control the same blot was also analyzed withantibodies directed against the platform protein LamB at a dilution of1:5,000 of hyperimmune rabbit serum. M, Molecular weight marker; L,Extract from a clone expressing LamB without foreign peptide insert.

FIG. 4(A) shows the representation of different serotypes of S.agalactiae clinical isolates analyzed for the gene distribution study. Anumber of the strains were not typeable and may represent additionalserotypes. (B) shows the PCR analysis for the gene distribution ofgbs0061 with the respective oligonucleotides and 46 S. agalactiaestrains. The predicted size of the PCR fragments is 814 bp. 1-46, S.agalactiae strains, clinical isolates as shown under A; −, no genomicDNA added; +, genomic DNA from S. agalactiae ATCC 12403, which served astemplate for library construction.

FIG. 5 shows the measurement of epitope-specific mouse serum IgGantibody levels induced by total bacterial lysates of Lamb or FhuAexpressing E. coli clones with S. agalactiae-derived epitopes. (A) showsrepresentative peptide ELISA experiments with three sets of mouse sera(5 mice in each group, 1-5) generated by gbs0428, gbs0628 and gbs632epitopes, respectively. Sera were tested at two different dilutions:black bars: 100×; grey bars; 1000×. Biotin-labeled synthetic peptidescorresponding to the respective epitopes were used in the peptide ELISA.Sera induced with E. coli lysate without S. agalactiae derived epitopesare indicated as FhuA or LamB. (B) shows a typical immunoblottingexperiment using lysates prepared from individual E. coli clonesselected for mouse injections. Sera were depleted by E. coli lysate notcarrying epitope to remove antibodies against E. coli proteins. Examplesare shown for gbs0918, gbs0428, gbs0628 and gbs632 epitopes. Negativecontrols (−) are E. coli clones with empty platform proteins. Locationof platform proteins LamB and FhuA is indicated by arrows.

FIG. 6 shows the detection of specific antibody binding on the cellsurface of Streptococcus agalactiae by flow cytometry. In FIG. 6Apreimmune mouse sera and polyclonal sera raised against S. agalactiaeserotype III lysate were incubated with S. agalactiae strain serotypeIII and analyzed by flow cytometry. Control shows the level ofnon-specific binding of the secondary antibody to the surface of S.agalactiae cells. The histograms in FIG. 6B indicates the increasedfluorescence due to specific binding of anti-gbs0031, anti-gbs1925 andanti-gbs0012 antibodies in comparison to the control sera generatedagainst E. coli lysate containing only the ‘empty’ platform proteinFhuA.

FIG. 7 shows the bactericidal activity of epitope specific antibodies asdetermined in in vitro killing assay. The killing activity of immunesera is measured parallel with and calculated relative to theappropriate control sera. Data are expressed as percentage of killing,that is the reduction on bacterial cfu numbers as a consequence of thepresence of specific antibodies. Hyperimmune polyclonal mouse seragenerated with S. agalactiae lysate and sera from non-immunized miceserved as positive and negative controls for the assay, respectively.Immune sera generated with (A) gbs0012, gbs0016, gbs0031, gbs0428,gbs1306 and gbs2018 epitopes and with (B) gbs0233, gbs0419, gbs0942,gbs0975, gbs1038, gbs1144 and gbs2093 epitopes were tested forbactericidal activity and data are expressed relative to appropriatecontrols, such as sera induced with Lamb or FhuA expressing E. coliclones without S. agalactiae-derived epitopes. S. agalactiae serotypeIII cells were incubated with mouse phagocytic cells for 60 min, andsurviving bacteria were quantified by counting cfus after plating onblood agar.

Table 1: Immunogenic Proteins Identified by Bacterial Surface Display.

(A) Columns A, 300 bp library of S. agalactiae ATCC 12403 in fhuA withNSag8-IgA (826), B, 300 bp library in fhuA with PSag10-IgA (768), C, 300bp library in fhuA with PSag10-IgG (711), D, 300 bp library in fhuA withPSag11-IgG (640), E, 70 bp library in lamB with NSag8-IgA (1057), F, 70bp library in lamB with NSag8-IgG (869), G, 70 bp library in lamB withPSag10-IgA (904), H, 70 bp library in lamB with PSag10-IgA-adsorbed(493), I, 70 bp library in lamB with PSag10-IgG (910), J, 70 bp libraryin lamB with PSag11-IgA (631), K, 70 bp library in lamB with PSag11-IgG(926), L, 70 bp library in lamB with PSag18-IgA (691), M, 70 bp libraryin lamB with PSag-sIgA (628); *, prediction of antigenic sequenceslonger than 5 amino acids was performed with the program ANTIGENIC(Kolaskar and Tongaonkar, 1990). Table 1B lists the immunogenic proteinsidentified by amino acid sequence identity with peptides identified bybacterial surface display. Antigenic peptides, which have beenidentified by bacterial surface-display possess identical counterpartsin the listed proteins from S. agalactiae. The peptides have been shownto react with multiple human sera (see Table 2). Sera directed againstthese peptides can therefore recognize multiple proteins.

Table 2: Epitope Serology with Human Sera.

Immune reactivity of individual synthetic peptides representing selectedepitopes with human sera is shown. Extent of reactivity is expressed as+, ++ or +++, and summed from individual reactivities of peptides withindividual sera (13 patient and 9 healthy adult, 22 total). A totalscore for each peptide was calculated based on ELISA units as the sum ofall reactivities. Scores were 2-8 for +, 9-16 for ++ and 17-26 for +++.ELISA units were calculated from OD_(405 nm) readings and the serumdilution after correction for background. Location of synthetic peptideswithin the antigenic ORFs according to the genome annotation of ATCC12403 strain is given in columns aa from and aa to indicating the firstand last amino acid residues, respectively. Peptide names: gbs0012.1present in annotated ORF: gbs0012.

Table 3: Gene distribution in S. agalactiae Strains.

Forty-six S. agalactiae strains as shown in FIG. 4A were tested by PCRwith oligonucleotides specific for the genes encoding relevant antigens.The PCR fragment of one selected PCR reaction was sequenced in order toconfirm the amplification of the correct DNA fragment. *, number ofamino acid substitutions in a serotype IA strain as derived fromsequencing as compared to S. agalactiae ATCC 12403. #, alternativestrain used for sequencing, because gene was not present in the serotypeIA strain.

Table 4: Immunogenicity of Antigenic Epitopes.

S. agalactiae antigens were tested for immunogenicity by immunizationwith E. coli clones harboring plasmids encoding the platform proteinsLamB or FhuA fused to S. agalactiae peptides. The presence ofepitope-specific antibodies were detected and measured by peptide ELISAand/or immunoblotting using the corresponding E. coli clone lysate,which served as immunogen. Results are expressed as + to ++++, andcalculated for peptide ELISA as the sum of the reactivity of individualmouse sera based on ELISA units (as indicated on FIG. 5A) and forimmunoblotting (IB) as the strength of reactivity of pooled (5individual) mouse sera with the epitope containing platform protein (asindicated on FIG. 5B). Location of synthetic peptides within theantigenic ORFs according to the genome annotation of ATCC 12403 strainis given in columns aa from and aa to indicating the first and lastamino acid residues, respectively.

Table 5: Immunogenic Proteins Identified by Bacterial Surface Display.

(A) 300 bp library of S. agalactiae ATCC 12403 in fhuA with IC8-IgA(826), B, 300 bp library in fhuA with P10-IgA (768), C, 300 bp libraryin fhuA with P10-IgG (711), D, 300 bp library in fhuA with P11-IgG(640), E, 70 bp library in lamB with IC8-IgA (1057), F, 70 bp library inlamB with IC8-IgG (869), G, 70bp library in lamB with P10-IgA (904), H,70 bp library in lamB with P10-IgA-adsorbed (493), I, 70 bp library inlamB with P10-IgG (910), J, 70 bp library in lamB with P11-IgA (631), K,70 bp library in lamB with P11-IgG (926), *, prediction of antigenicsequences longer than 5 amino acids was performed with the programANTIGENIC (Kolaskar and Tongaonkar, 1990).

Table 6: Epitope Eerology with Human Sera.

Immune reactivity of individual synthetic peptides representing selectedepitopes with human sera is shown. Extent of reactivity is expressed as+, ++ or +++, and summed from individual reactivities of peptides withindividual sera (13 patient and 9 healthy adult, 22 total). A totalscore for each peptide was calculated based on ELISA units as the sum ofall reactivities. Scores were 2-8 for +, 9-16 for ++ and 17-30 for +++.ELISA units were calculated from OD_(405 nm) readings and the serumdilution after correction for background. Location of synthetic peptideswithin the antigenic ORFs according to the genome annotation of ATCC12403 strain is given in columns aa from and aa to indicating the firstand last amino acid residues, respectively. Peptide names: gbs0233.1present in annotated ORF: gbs0233.

Table 7: Immunogenicity of Antigenic Epitopes in Mice.

S. agalactiae antigens were tested for immunogenicity by immunizationwith E. coli clones harboring plasmids encoding the platform proteinsLamB or FhuA fused to S. agalactiae peptides. The presence ofepitope-specific antibodies were detected and measured by peptide ELISA.Results are expressed as + to +++++, and calculated for peptide ELISA asthe sum of the reactivity of individual mouse sera based on ELISA units(as indicated on FIG. 5). Location of epitopes within the antigenic ORFsaccording to the genome annotation of ATCC 12403 strain is given incolumns aa from and aa to indicating the first and last amino acidresidues, respectively.

EXAMPLES Example 1 Characterization and Selection of human Serum SourcesBased on Anti-S. agalactiae Antibodies, Preparation of AntibodyScreening Reagents

Experimental Procedures

Enzyme Linked Immune Assay (ELISA).

ELISA plates (Maxisorb, Millipore) were coated with 5-10 μg/ml totalprotein diluted in coating buffer (0.1M sodium carbonate pH 9.2). Threedilutions of sera (2,000×, 10,000×, 50,000×) were made in PBS-BSA.Highly specific Horse Radish Peroxidase (HRP)-conjugated anti-human IgGor anti-human IgA secondary antibodies (Southern Biotech) were usedaccording to the manufacturers' recommendations (dilution: 1,000×).Antigen-antibody complexes were quantified by measuring the conversionof the substrate (ABTS) to colored product based on OD_(405 nm) readingsby automatic ELIAS reader (TECAN SUNRISE).

Preparation of Bacterial Antigen Extracts

Total bacterial lysate: Bacteria were grown overnight in THB(Todd-Hewitt Broth) and lysed by repeated freeze-thaw cycles: incubationon dry ice/ethanol-mixture until frozen (1 min), then thawed at 37°0 C.(5 min): repeated 3 times. This was followed by sonication andcollection of supernatant by centrifugation (3,500 rpm, 15 min, 4°0 C.).

Culture supernatant: After removal of bacteria by centrifugation, thesupernatant of overnight grown bacterial cultures was precipitated withice-cold ethanol by mixing 1 part supernatant with 3 parts absoluteethanol and incubated overnight at −20° C. Precipitates were collectedby centrifugation (2,600 g, for 15 min). Dry pellets were dissolvedeither in PBS for ELISA, or in urea and SDS-sample buffer for SDS-PAGEand immunoblotting. The protein concentration of samples was determinedby Bradford assay.

Immunoblotting

Total bacterial lysate and culture supernatant samples were preparedfrom in vitro grown S. agalactiae serotype III strain. 10 to 25 μg totalprotein/lane was separated by SDS-PAGE using the BioRad Mini-ProteanCell electrophoresis system and proteins transferred to nitrocellulosemembrane (ECL, Amersham Pharmacia). After overnight blocking in 5% milk,human sera were added at 2,000× dilution, and HRPO labeled anti-humanIgG was used for detection.

Extraction of Antibodies From Cervical Wicks

Cervical secretions were collected by absorbent cylindrical wicks(Polyfiltronics) which were introduced into the cervical canal duringspeculum examination and thereafter kept frozen until extraction.Extraction was done according to Hordnes et al, 1998 (provider of thesamples). Briefly, wicks were mixed with PBS containing proteaseinhibitors, vortexed and fluid was drained from the tubes containing thewicks. The concentrations of total IgA and IgG antibodies in extractswere determined.

Purification of antibodies for genomic screening. Five sera from boththe patient and the healthy group were selected based on the overallanti-GBS titers for serum or cervical secretion pools used in thescreening procedure. Antibodies against E. coli proteins were removed byincubating the heat-inactivated sera with whole cell E. coli cells(DH5alpha, transformed with pHlE11, grown under the same condition asused for bacterial surface display). Highly enriched preparations ofIgGs from the pooled, depleted sera were generated by protein G affinitychromatography, according to the manufacturer's instructions (UltraLinkImmobilized Protein G, Pierce). IgA antibodies were purified also byaffinity chromatography using biotin-labeled anti-human IgA (SouthernBiotech) immobilized on Streptavidin-agarose (GIBCO BRL). The efficiencyof depletion and purification was checked by SDS-PAGE, Western blotting,ELISA and protein concentration measurements.

Results

The antibodies produced against S. agalactiae by the human immune systemand present in human sera are indicative of the in vivo expression ofthe antigenic proteins and their immunogenicity. These molecules areessential for the identification of individual antigens in the approachas described in the present invention, which is based on the interactionof the specific anti-GBS antibodies and the corresponding S. agalactiaepeptides or proteins. To gain access to relevant antibody repertoires,human sera were collected from

-   -   I. healthy pregnant women tested negative for cervical and        anorectal carriage of GBS    -   II. healthy pregnant women tested positive for cervical and/or        anorectal carriage of GBS who's newborn remained GBS-free        (although with antibiotic prevention).    -   III. adults below <45 years of age without clinical disease.    -   IV. naïve individuals, young children between 5 and 10 months of        age, after they already lost maternal antibodies and have not        acquired GBS-specific ones due to the lack of GBS disease.

In addition cervical secretions were also collected from the first twogroups of donors. The extreme value of these antibody sources is mainlythe secretory IgA component, which is directly implicated as protectiveeffector molecule on mucosal surfaces.

It is important to screen with antibodies from at least two differentpopulations, pregnant women and nonpregnant adults, since GBS diseaseaffects elderly and immunocompromised adults, as well. Within thepregnant study group, there are again two different patient categories,women who are GBS colonized and those who are noncolonized, to beincluded in the antigen screen.

Antibodies in serum and other body fluids, such as mucosal secretionsinduced in individuals exposed to the pathogens are crucial for antigenidentification. The exposure to GBS results in asymptomaticcolonization, current or past acute or chronic infection. S. agalactiaecolonization and infections are common, and antibodies are present as aconsequence of natural immunization from previous encounters. It islikely that sera from high titer noncolonized individuals containfunctional antibodies, which are able to eliminate carriage. At the sametime certain antibodies might be induced against GBS components only ifthe antigen persist. For that reason sera from colonized individualswere also included. It has been shown that colonization is associatedwith capsular polysaccharide (CPS)-specific antibody responses. However,it is not clear whether sufficient level of antibodies to CPS's wouldprevent GBS colonization, since there are colonized women with both highand low levels of anti-CPS antibody, and the same is true fornoncolonized pregnant women.

However, there are reports that effector function and avidity ofantibodies produced during pregnancy might be altered. It is importantto recognize that most healthy adults are protected from invasive GBSdisease and are less susceptible than newborns and the elderly.Antibodies from these individuals seem to be especially valuable foridentification of the corresponding antigens. It is known that anti-GBSantibody levels increase with age.

GBS is a mucosal pathogen and should induce IgA response; for thatreason it was important to perform IgA-based screens, as well asIgG-based screens. The fact that some S. agalactiae strains express highaffinity IgA-binding receptor also points to the importance of IgA inhost response. Recently it was reported that not only IgG, but also IgAserum antibodies can be recognized by the FcRIII receptors of PMNs andpromote opsonization {Phillips-Quagliata, J. et al., 2000}; {Shibuya, A.et al., 2000}. The primary role of IgA antibodies is neutralization,mainly at the mucosal surface. The level of serum IgA reflects thequality, quantity and specificity of the dimeric secretory IgA. For thatreason the serum collection was not only analyzed for anti-streptococcalIgG, but also for IgA levels. In the ELISA assays highly specificsecondary reagents were used to detect antibodies from the high affinitytypes, such as IgG and IgA, but avoided IgM. Production of IgMantibodies occurs during the primary adaptive humoral response, andresults in low affinity antibodies, while IgG and IgA antibodies hadalready undergone affinity maturation, and are more valuable in fightingor preventing disease.

127 serum samples and 97 cervical secretions from pregnant women and 50sera from healthy adults were characterized for anti-S. agalactiaeantibodies by a series of immune assays. Primary characterization wasdone by ELISA using two different antigen preparations, such as totalbacterial extract and culture supernatant proteins prepared from S.agalactiae serotype III ATCC 12403 strain. A representative experimentis shown in FIG. 1A using sera from the healthy adult population.Antibody titers were compared at given dilutions where the response waslinear. Sera were ranked based on the IgG and IgA reactivity against thetwo complex antigenic mixtures (including serotype specific type IIIcapsule), and the highest ones were selected for further testing byimmunoblotting. This analysis confirmed a high antibody reactivity ofthe pre-selected sera against multiple GBS proteins, especially whencompared to not selected, low-titer sera (FIG. 1B). However, ELISAranking of sera did not always correlated with immunoblot signalssuggesting that anti-capsular antibodies were abundant and dominated theELISA reactivities against total bacterial extracts. Thus the finalselection of sera to be included in antibody-pools was based mainly onmultiple immunogenic bands in immunoblotting experiments. This extensiveantibody characterization approach has led to the unambiguousidentification of anti-GBS hyperimmune sera.

The 97 cervical secretions were determined for IgA content, and sameamount (2 μg) was tested for anti-GBS reactivity by immunoblotting.Positively selected sera (as it is shown in FIG. 1C) were divided intocolonized and noncolonized IgA pools and used separately in bacterialsurface display experiments.

5 sera from both donor groups were selected and pooled for antigenidentification by bacterial surface display. Selected sera included inthe four pregnant women pools (PSAg10-IgG,-IgA, PSAg11-IgG, PSAg18-IgGand PSAg-sIgA) and one healthy adult (non-pregnant) pool(NSAg8-IgG,-IgA). IgG and IgA antibodies were purified from pooled seraby affinity chromatography and depleted of E. coli-reactive antibodiesto avoid background in the bacterial surface display screen.

Example 2 Generation of Highly Random, Frame-selected, Small-fragment,Genomic DNA Libraries of Streptococcus agalactiae

Experimental Procedures

Preparation of streptococcal genomic DNA. 50 ml Todd-Hewitt Broth mediumwas inoculated with S. agalactiae ATCC 12403 bacteria from a frozen staband grown with aeration and shaking for 18 h at 37° C. The culture wasthen harvested, centrifuged with 1,600×g for 15 min and the supernatantwas removed. Bacterial pellets were washed 3× with PBS and carefullyre-suspended in 0.5 ml of Lysozyme solution (100 mg/ml). 0.1 ml of 10mg/ml heat treated RNase A and 20 U of RNase T1 were added, mixedcarefully and the solution was incubated for 1 h at 37° C. Following theaddition of 0.2 ml of 20% SDS solution and 0.1 ml of Proteinase K (10mg/ml) the tube was incubated overnight at 55° C. ⅓ volume of saturatedNaCl was then added and the solution was incubated for 20 min at 4° C.The extract was pelleted in a microfuge (13,000 rpm) and the supernatanttransferred into a new tube. The solution was extracted withPhOH/CHCl₃/IAA (25:24:1) and with CHCl₃/IAA (24:1). DNA was precipitatedat room temperature by adding 0.6× volume of Isopropanol, spooled fromthe solution with a sterile Pasteur pipette and transferred into tubescontaining 80% ice-cold ethanol. DNA was recovered by centrifuging theprecipitates with 10-12,000×g, then dried on air and dissolved in ddH₂O.

Preparation of small genomic DNA fragments. Genomic DNA fragments weremechanically sheared into fragments ranging in size between 150 and 300bp using a cup-horn sonicator (Bandelin Sonoplus UV 2200 sonicatorequipped with a BB5 cup horn; 10 sec. pulses at 100% power output) orinto fragments of size between 50 and 70 bp by mild DNase I treatment(Novagen). It was observed that sonication yielded a much tighterfragment size distribution when breaking the DNA into fragments of the150-300 bp size range. However, despite extensive exposure of the DNA toultrasonic wave-induced hydromechanical shearing force, subsequentdecrease in fragment size could not be efficiently and reproduciblyachieved. Therefore, fragments of 50 to 70 bp in size were obtained bymild DNase I treatment using Novagen's shotgun cleavage kit. A 1:20dilution of DNase I provided with the kit was prepared and the digestionwas performed in the presence of MnCl₂ in a 60 μl volume at 20° C. for 5min to ensure double-stranded cleavage by the enzyme. Reactions werestopped with 2 μl of 0.5 M EDTA and the fragmentation efficiency wasevaluated on a 2% TAE-agarose gel. This treatment resulted in totalfragmentation of genomic DNA into near 50-70 bp fragments. Fragmentswere then blunt-ended twice using T4 DNA Polymerase in the presence of100 μM each of dNTPs to ensure efficient flushing of the ends. Fragmentswere used immediately in ligation reactions or frozen at −20° C. forsubsequent use.

Description of the vectors. The vector pMAL4.31 was constructed on apASK-IBA backbone {Skerra, A., 1994} with the beta-lactamase (bla) geneexchanged with the Kanamycin resistance gene. In addition the bla genewas cloned into the multiple cloning site. The sequence encoding maturebeta-lactamase is preceded by the leader peptide sequence of ompA toallow efficient secretion across the cytoplasmic membrane. Furthermore asequence encoding the first 12 amino acids (spacer sequence) of maturebeta-lactamase follows the ompA leader peptide sequence to avoid fusionof sequences immediately after the leader peptidase cleavage site, sincee.g. clusters of positive charged amino acids in this region woulddecrease or abolish translocation across the cytoplasmic membrane{Kajava, A. et al., 2000}. A SmaI restriction site serves for libraryinsertion. An upstream FseI site and a downstream NotI site, which wereused for recovery of the selected fragment, flank the SmaI site. Thethree restriction sites are inserted after the sequence encoding the 12amino acid spacer sequence in such a way that the bla gene istranscribed in the −1 reading frame resulting in a stop codon 15 bpafter the NotI site. A +1 bp insertion restores the bla ORF so thatbeta-lactamase protein is produced with a consequent gain of Ampicillinresistance.

The vector pMAL9.1 was constructed by cloning the lamB gene into themultiple cloning site of pEH1 {Hashemzadeh-Bonehi, L. et al., 1998}.Subsequently, a sequence was inserted in lamB after amino acid 154,containing the restriction sites FseI, SmaI and NotI. The reading framefor this insertion was constructed in such a way that transfer offrame-selected DNA fragments excised by digestion with FseI and NotIfrom plasmid pMAL4.31 yields a continuous reading frame of lamB and therespective insert.

The vector pHIE11 was constructed by cloning the fhuA gene into themultiple cloning site of pEH1. Thereafter, a sequence was inserted infhuA after amino acid 405, containing the restriction site FseI, XbaIand NotI. The reading frame for this insertion was chosen in a way thattransfer of frame-selected DNA fragments excised by digestion with FseIand NotI from plasmid pMAL4.31 yields a continuous reading frame of fhuAand the respective insert.

Cloning and evaluation of the library for frame selection. Genomic S.agalactiae DNA fragments were ligated into the SmaI site of the vectorpMAL4.31. Recombinant DNA was electroporated into DH10B electrocompetentE. coli cells (GIBCO BRL) and transformants plated on LB-agarsupplemented with Kanamycin (50 μg/ml) and Ampicillin (50 μg/ml). Plateswere incubated over night at 37° C. and colonies collected for largescale DNA extraction. A representative plate was stored and saved forcollecting colonies for colony PCR analysis and large-scale sequencing.A simple colony PCR assay was used to initially determine the roughfragment size distribution as well as insertion efficiency. Fromsequencing data the precise fragment size was evaluated, junctionintactness at the insertion site as well as the frame selection accuracy(3n+1 rule).

Cloning and evaluation of the library for bacterial surface display.Genomic DNA fragments were excised from the pMAL4.31 vector, containingthe S. agalactiae library with the restriction enzymes FseI and NotI.The entire population of fragments was then transferred into plasmidspMAL9.1 (LamB) or pHIE11 (FhuA), which have been digested with FseI andNotI. Using these two restriction enzymes, which recognise an 8 bp GCrich sequence, the reading frame that was selected in the pMAL4.31vector is maintained in each of the platform vectors. The plasmidlibrary was then transformed into E. coli DH5alpha cells byelectroporation. Cells were plated onto large LB-agar platessupplemented with 50 μg/ml Kanamycin and grown over night at 37° C. at adensity yielding clearly visible single colonies. Cells were thenscraped off the surface of these plates, washed with fresh LB medium andstored in aliquots for library screening at −80° C.

Results

Libraries for frame selection. Two libraries (LSAg-70 and LSAg-300) weregenerated in the pMAL4.31 vector with sizes of approximately 70 and 300bp, respectively. For each library, ligation and subsequenttransformation of approximately 1 μg of pMAL4.31 plasmid DNA and 50 ngof fragmented genomic S. agalactiae DNA yielded 4×10⁵ to 2×10⁶ clonesafter frame selection. To assess the randomness of the libraries,approximately 576 randomly chosen clones of LSAg-70 were sequenced. Thebioinformatic analysis showed that of these clones only very few werepresent more than once. Furthermore, it was shown that approximately 80%of the clones fell in the size range between 25 and 100 bp with anaverage size of approximately 40 bp (FIG. 2). Almost all sequencesfollowed the 3n+1 rule, showing that all clones were properly frameselected.

Bacterial surface display libraries. The display of peptides on thesurface of E. coli required the transfer of the inserts from the LSAg-70and LSAg-300 libraries from the frame selection vector pMAL4.31 to thedisplay plasmids pMAL9.1 (LamB) or pHIE11 (FhuA). Genomic DNA fragmentswere excised by FseI and NotI restriction and ligation of 5 ng insertswith 0.1 μg plasmid DNA and subsequent transformation into DH5alphacells resulted in 2-5×10⁶ clones. The clones were scraped off the LBplates and frozen without further amplification.

Example 3 Identification of Highly Immunogenic Peptide Sequences from S.agalactiae Using Bacterial Surface Displayed Genomic Libraries and HumanSerum

Experimental Procedures

MACS screening. Approximately 2.5×10⁸ cells from a given library weregrown in 5 ml LB-medium supplemented with 50 μg/ml Kanamycin for 2 h at37° C. Expression was induced by the addition of 1 mM IPTG for 30 min.Cells were washed twice with fresh LB medium and approximately 2×10⁷cells re-suspended in 100 μl LB medium and transferred to an Eppendorftube.

10 to 20 μg of biotinylated, human IgGs purified from serum was added tothe cells and the suspension incubated overnight at 4° C. with gentleshaking. 900 μl of LB medium was added, the suspension mixed andsubsequently centrifuged for 10 min at 6,000 rpm at 4° C. (For IgAscreens, 10 μg of purified IgAs were used and these captured withbiotinylated anti-human-IgG secondary antibodies). Cells were washedonce with 1 ml LB and then re-suspended in 100 μl LB medium. 10 μl ofMACS microbeads coupled to streptavidin (Miltenyi Biotech, Germany) wereadded and the incubation continued for 20 min at 4° C. Thereafter 900 μlof LB medium was added and the MACS microbead cell suspension was loadedonto the equilibrated MS column (Miltenyi Biotech, Germany) which wasfixed to the magnet. (The MS columns were equilibrated by washing oncewith 1 ml 70% EtOH and twice with 2 ml LB medium.)

The column was then washed three times with 3 ml LB medium. Afterremoval of the magnet, cells were eluted by washing with 2 ml LB medium.After washing the column with 3 ml LB medium, the 2 ml eluate was loadeda second time on the same column and the washing and elution processrepeated. The loading, washing and elution process was performed a thirdtime, resulting in a final eluate of 2 ml.

A second and third round of screening was performed as follows. Thecells from the final eluate were collected by centrifugation andre-suspended in 1 ml LB medium supplemented with 50 μg/ml Kanamycin. Theculture was incubated at 37° C. for 90 min and then induced with 1 mMIPTG for 30 min. Cells were subsequently collected, washed once with 1ml LB medium and suspended in 10 μl LB medium. 10 to 20 μg of human,biotinylated IgGs were added again and the suspension incubated overnight at 4° C. with gentle shaking. All further steps were exactly thesame as in the first selection round. Cells selected after two rounds ofselection were plated onto LB-agar plates supplemented with 50 μg/mlKanamycin and grown over night at 37° C.

Evaluation of selected clones by sequencing and Western blot analysis.Selected clones were grown overnight at 37° C. in 3 ml LB mediumsupplemented with 50 μg/ml Kanamycin to prepare plasmid DNA usingstandard procedures. Sequencing was performed at MWG (Germany) or incollaboration with TIGR (U.S.A.).

For Western blot analysis approximately 10 to 20 μg of total cellularprotein was separated by 10% SDS-PAGE and blotted onto HybondC membrane(Amersham Pharmacia Biotech, England). The LamB or FhuA fusion proteinswere detected using human serum as the primary antibody at a dilution ofapproximately 1:3,000 to 1:5,000 and anti-human IgG or IgA antibodiescoupled to HRP at a dilution of 1:5,000 as secondary antibodies.Detection was performed using the ECL detection kit (Amersham PharmaciaBiotech, England). Alternatively, rabbit anti-FhuA or rabbit anti-LamBpolyclonal immune sera were used as primary antibodies in combinationwith the respective secondary antibodies coupled to HRP for thedetection of the fusion proteins.

Results

Screening of bacterial surface display libraries by magnetic activatedcell sorting (MACS) using biotinylated Igs. The libraries LSag-70 inpMAL9.1 and LSag-300 in pHIE11 were screened with pools of biotinylated,human IgGs and IgAs prepared from sera of healthy adults(NSag8-IgG,-IgA) or P10,11,18 (see Example 1: Preparation of antibodiesfrom human serum). The selection procedure was performed as describedunder Experimental procedures. FIG. 3A shows a representative example ofa screen with the LSag-70 library and PSag11-IgGs. As can be seen fromthe colony count after the first selection cycle from MACS screening,the total number of cells recovered at the end is drastically reducedfrom 2×10⁷ cells to approximately 2×10⁴ cells, but the selection withoutantibodies added showed a similar reduction in cell numbers (FIG. 3A).Therefore a second and third round of selection was performed. At theend of round three, approximately 10⁴ cells was recovered withPSag11-IgGs, while only 2×10³ cells were recovered when no IgGs fromhuman serum were added, clearly showing that selection was dependent onS. agalactiae specific antibodies. To evaluate the performance of thescreen, 26 selected clones were picked randomly and subjected toimmunoblot analysis with screening IgG pool (FIG. 3B). This analysisrevealed that more than 80% of the selected clones showed reactivitywith antibodies present in the relevant serum whereas the control strainexpressing LamB without a S. agalactiae specific insert did not reactwith the same serum. In general, the rate of reactivity was observed tolie within the range of 35 to 90%. Colony PCR analysis showed that allselected clones contained an insert in the expected size range.

Subsequent sequencing of a larger number of randomly picked clones (600to 1200 per screen) led to the identification of the gene and thecorresponding peptide or protein sequence that was specificallyrecognized by the human serum antibodies used for screening. Thefrequency with which a specific clone is selected reflects at least inpart the abundance and/or affinity of the specific antibodies in theserum used for selection and recognizing the epitope presented by thisclone. In that regard it is striking that clones derived from some ORFs(e.g. gbs1087, gbs1306, gbs2018) were picked more than 100 times,indicating their highly immunogenic property. Table 1 and Table 5summarizes the data obtained for all 13 performed screens. All clonesthat are presented in Table 1 and Table 5 have been verified byimmunoblot analysis using whole cellular extracts from single clones toshow the indicated reactivity with the pool of human serum used in therespective screen. As can be seen from Table 1 and Table 5, distinctregions of the identified ORF are identified as immunogenic, sincevariably sized fragments of the proteins are displayed on the surface bythe platform proteins.

It is further worth noticing that most of the genes identified by thebacterial surface display screen encode proteins that are eitherattached to the surface of S. agalactiae and/or are secreted. This is inaccordance with the expected role of surface attached or secretedproteins in virulence of S. agalactiae.

Example 4

Assessment of the Reactivity of Highly Immunogenic Peptide Sequenceswith Individual Human Sera.

Experimental Procedures

Peptide Synthesis

Peptides were synthesized in small scale (4 mg resin; up to 288 inparallel) using standard F-moc chemistry on a Rink amide resin (PepChem,Tübingen, Germany) using a SyroII synthesizer (Multisyntech, Witten,Germany). After the sequence was assembled, peptides were elongated withFmoc-epsilon-aminohexanoic acid (as a linker) and biotin (Sigma, St.Louis, Mo.; activated like a normal amino acid). Peptides were cleavedoff the resin with 93% TFA, 5% triethylsilane, and 2% water for onehour. Peptides were dried under vacuum and freeze dried three times fromacetonitrile/water (1:1). The presence of the correct mass was verifiedby mass spectrometry on a Reflex III MALDI-TOF (Bruker, Bremen Germany).The peptides were used without further purification.

Enzyme Linked Immune Assay (ELISA).

Biotin-labeled peptides (at the N-terminus) were coated on StreptavidinELISA plates (EXICON) at 10 μg/ml concentration according to themanufacturer's instructions. Highly specific Horse Radish Peroxidase(HRP)-conjugated anti-human IgG secondary antibodies (Southern Biotech)were used according to the manufacturers' recommendations (dilution:1,000×). Sera were tested at two serum dilutions, 200× and 1,000×.Following manual coating, peptide plates were processed and analyzed bythe Gemini 160 ELISA robot (TECAN) with a built-in ELISA reader (GENIOS,TECAN).

Results

Following the bioinformatic analysis of selected clones, correspondingpeptides were designed and synthesized. In case of epitopes with morethan 26 amino acid residues, overlapping peptides were made. Allpeptides were synthesized with a N-terminal biotin-tag and used ascoating reagents on Streptavidin-coated ELISA plates.

The analysis was performed with peptides that were selected based ontheir reactivity with the individual sera, which were included in theserum pools used for preparations of IgG and IgA screening reagents forbacterial surface display. A summary for serum reactivity of 74 peptidesrepresenting 55 different S. agalactiae antigenic proteins from thegenomic screen analyzed with 22 human sera (from 13 patient and 9healthy exposed high titer individuals) used for the antigenidentification is shown in Table 2 and Table 6. The peptides werecompared by the score calculated for each peptide based on the number ofpositive sera and the extent of reactivity. Peptides range from highlyand widely reactive to weakly positive ones.

Example 5 Gene Distribution Studies with Highly Immunogenic ProteinsIdentified from S. agalactiae.

Experimental Procedures

Gene distribution of GBS antigens by PCR. An ideal vaccine antigen wouldbe an antigen that is present in all, or the vast majority of strains ofthe target organism to which the vaccine is directed. In order toestablish whether the genes encoding the identified Streptococcusagalactiae antigens occur ubiquitously in S. agalactiae strains, PCR wasperformed on a series of independent S. agalactiae isolates with primersspecific for the gene of interest. S. agalactiae isolates were obtainedcovering the serotypes most frequently present in patients as shown inFIG. 4A. Oligonucleotide sequences as primers were designed for allidentified ORFs yielding products of approximately 1,000 bp, if possiblecovering all identified immunogenic epitopes. Genomic DNA of all S.agalactiae strains was prepared as described under Example 2. PCR wasperformed in a reaction volume of 25 μl using Taq polymerase (1U), 200nM dNTPs, 10 pMol of each oligonucleotide and the kit according to themanufacturers instructions (Invitrogen, The Netherlands). As standard,30 cycles (1×: 5 min. 95° C., 30×: 30 sec. 95° C., 30 sec. 56° C., 30sec. 72° C., 1×4 min. 72° C.) were performed, unless conditions had tobe adapted for individual primer pairs.

Results

Identified genes encoding immunogenic proteins were tested by PCR fortheir presence in 46 different strains of S. agalactiae (FIG. 4A). As anexample, FIG. 4B shows the PCR reaction for GBS0061 with all indicated46 strains. As clearly visible, the gene is present in all strainsanalyzed. The PCR fragment from a type IA strain was sequenced andshowed that all 657 bp were identical as compared to the S. agalactiaeATCC 12403 strain, indicating a high level of conservation between thetwo isolates. From a total of 117 genes analyzed, more than 100 werepresent in all or almost all strains tested, while only 5 genes wereabsent in more than 10% of the tested 46 strains (Table 3). In addition,only few genes (e.g. GBS0016, GBS1087, GBS1528 and GBS2018) showedvariation in size but were present in all or most strain isolates.Sequencing of the generated PCR fragment from one strain and subsequentcomparison to the type III strain ATCC 12403 confirmed the amplificationof the correct DNA fragment and revealed a degree of sequence divergenceas indicated in Table 3. Importantly, many of the identified antigensare well conserved in all strains in sequence and size and are thereforenovel vaccine candidates to prevent infections by GBS.

Example 6 Characterization of Immune Sera Obtained from Mice Immunizedwith Highly Immunogenic Proteins/peptides from S. agalactiae Displayedon the Surface of E. coli.

Experimental Procedures

Generation of Immune Sera from Mice

E. coli clones harboring plasmids encoding the platform protein fused toa S. agalactiae peptide, were grown in LB medium supplemented with 50μg/ml Kanamycin at 37° C. Overnight cultures were diluted 1:10, grownuntil an OD₆₀₀ of 0.5 and induced with 0.2 mM IPTG for 2 hours. Pelletedbacterial cells were suspended in PBS buffer and disrupted by sonicationon ice, generating a crude cell extract. According to the OD₆₀₀measurement, an aliquot corresponding to 5×10⁷ cells was injected intoNMRI mice i.v., followed by a boost after 2 weeks. Serum was taken 1week after the second injection. Epitope specific antibody levels weremeasured by peptide ELISA.

In vitro Expression of Antigens

Expression of antigens by in vitro grown S. agalactiae serotype III wastested by immunoblotting. Different growth media and culture conditionswere tested to detect the presence of antigens in total lysates andbacterial culture supernatants. Expression was considered confirmed whena specific band corresponding to the predicted molecular weight andelectrophoretic mobility was detected. ps Cell Surface Staining

Flow cytometric analysis was carried out as follows. Bacteria were grownunder culture conditions, which resulted in expression of the antigen asshown by the immunoblot analysis. Cells were washed twice in HanksBalanced Salt Solution (HBSS) and the cell density was adjusted toapproximately 1×10⁶ CFU in 100 μl HBSS, 0.5% BSA. After incubation for30 to 60 min at 4° C. with mouse antisera diluted 50 to 100-fold,unbound antibodies were washed away by centrifugation in excess HBSS,0.5% BSA. Secondary goat anti-mouse antibody (F(ab′)₂ fragment specific)labeled with fluorescein (FITC) was incubated with the cells at 4° C.for 30 to 60 min. After washing, cells were fixed with 2%paraformaldehyde. Bound antibodies were detected using a BectonDickinson FACScan flow cytometer and data further analyzed with thecomputer program CELLQuest. Negative control sera included mousepre-immune serum and mouse polyclonal serum generated with lysatesprepared from IPTG induced E. coli cells transformed with plasmidsencoding the genes lamB or fhuA without S. agalactiae genomic insert.

Bactericidal (Killing) Assay

Murine macrophage cells (RAW246.7 or P388.D1) and bacteria wereincubated and the loss of viable bacteria after 60 min was determined bycolony counting. In brief, bacteria were washed twice in Hanks BalancedSalt Solution (HBSS) and the cell density was adjusted to approximately1×10⁵ CFU in 50 μl HBSS. Bacteria were incubated with mouse sera (up to25%) and guinea pig complement (up to 5%) in a total volume of 100 μlfor 60 min at 4° C. Pre-opsonized bacteria were mixed with macrophages(murine cell line RAW264.7 or P388.D1; 2×10⁶ cells per 100 μl) at a 1:20ratio and were incubated at 37° C. on a rotating shaker at 500 rpm. Analiquot of each sample was diluted in sterile water and incubated for 5min at room temperature to lyse macrophages. Serial dilutions were thenplated onto Todd-Hewitt Broth agar plates. The plates were incubatedovernight at 37° C., and the colonies were counted with the Countermatflash colony counter (IUL Instruments). Control sera included mousepre-immune serum and mouse polyclonal serum generated with lysatesprepared from IPTG induced E. coli transformed with plasmids harboringthe genes lamB or fhuA without S. agalactiae genomic insert.

Results

Immunogenicity in mice. The presence of specific antibodies wasdetermined by peptide ELISA and/or immunoblotting using the E. coliclone expressing the given epitope embedded in LamB or FhuA platformproteins, as it is exemplified in FIG. 5A and B, respectively, andsummarized in Table 4 and Table 7. 43 novel GBS antigens represented by61 different epitope regions were shown to be immunogenic in mice.Positive sera were then analyzed by immunoblotting using total bacteriallysates and culture supernatants prepared from S. agalactiae serotypeIII strain (data not shown). This analysis served as a first step todetermine whether the antigenic proteins were expressed, and if, underwhich growth conditions, in order to evaluate surface expression of thepolypeptide by FACS analysis. It was anticipated based on literaturedata that not all proteins would be expressed under in vitro conditions.

Cell surface staining of S. agalactiae. Cell surface accessibility forseveral antigenic proteins was subsequently demonstrated by an assaybased on flow cytometry. GBS cells were incubated with preimmune andpolyclonal mouse sera raised against S. agalactiae lysate or E. coliclones harboring plasmids encoding the platform protein fused to a S.agalactiae peptide, followed by detection with fluorescently taggedsecondary antibody. As shown in FIG. 6A, antisera raised against S.agalactiae lysate contains antibodies against surface components,demonstrated by a significant shift in fluorescence of the S. agalactiaeserotype III cell population. Similar cell surface staining of S.agalactiae serotype III cells was observed with polyclonal sera raisedagainst peptides of many of the GBS antigens identified (FIG. 6B ). Insome instances, a subpopulation of the bacteria was not stained, asindicated by the detection of two peaks in the histograms (FIG. 6B).This phenomenon may be a result of differential expression of the geneproducts during the growth of the bacterium, insufficient antibodylevels or partial inhibition of antibody binding caused by other surfacemolecules or plasma proteins. Importantly, a well-known protective GBSantigen, Sip/gbs0031 is proved to be also positive in this assay.

In vitro bactericidal activity. Opsonophagocytic killing is thecornerstone of host defense against extracellular bacteria, such as S.agalactiae. Cell surface binding of antibodies to bacterial antigens areopsonizing and induce killing (bactericidal) by phagocytic cells(macrophages and neutrophil granulocytes) if the antibodies induced bythe particular antigens can bind activated complement components (C3bi).In FIG. 7 data are presented on bactericidal activity measured byantigen-specific antibodies generated in mice with correspondingepitopes. According to these data, several of the novel GBS antigens,for example gbs0012, gbs0016, gbs0428, gbs1306 and gbs2018 inducefunctional antibodies. Importantly, a well-known protective GBS antigen,Sip/gbs0031 is proved to be strongly positive in the very same assay.

These experiments confirmed the bioinformatic prediction that many ofthe proteins are exported due to their signal peptide sequence and inaddition showed that they are present on the cell surface of S.agalactiae serotype III. They also confirm that these proteins areavailable for recognition by human antibodies with functional propertiesand make them valuable candidates for the development of a vaccineagainst GBS diseases.

TABLE 1A Immunogenic proteins identified by bacterial surface display.S. agalactiae Location of identified antigenic No. of selected clonesper ORF immunogenic protein Putative function (by homology) predictedimmunogenic aa** and screen region (aa) Seq. ID (DNA, Prot.) gbs0012weakly similar to 4–20, 35–44, 65–70, 73–87, 92–98, 112–137, 152–161,177–186, B: 13, C: 6 115–199 1, 218 beta-lactamase 193–200, 206–213,229–255, 282–294, 308–313, 320–326, 349–355, 373–384, 388–406, 420–425gbs0016 glucan-binding 5–24, 35–41, 44–70, 73–89, 103–109, 127–143,155–161, 185–190, B: 12, C: 4, 1–75, 76–161, 2, 219 protein B 192–207,212–219, 246–262, 304–336, 372–382, 384–393, D: 3, E: 5, H: 4, 164–239398–407, 412–418, 438–444 I: 12, M: 2 gbs0024 phosphoribosylformyl 4–10,16–58, 60–71, 77–92, 100–126, 132–146, 149–164, 166–172, F: 4 802–812 3,220 glycinamidine 190–209, 214–220, 223–229, 241–256, 297–312, 314–319,synthase 337–343, 351–359, 378–387, 398–418, 421–428, 430–437, 440–448,462–471, 510–519, 525–536, 552–559, 561–568, 573–582, 596–602, 608–630,637–649, 651–665, 681–702, 714–732, 739–745, 757–778, 790–805, 807–815,821–829, 836–842, 846–873, 880–903, 908–914, 916–923, 931–940, 943–948,956–970, 975–986, 996–1015, 1031–1040, 1051–1069, 1072–1095, 1114–1119,1130–1148, 1150–1157, 1169–1176, 1229–1238 gbs0031 surface immunogenic5–12, 14–26, 35–47, 52–67, 72–78, 83–98, 121–141, 152–159, 163–183, A:17, B: 53, 46–291 4, 221 protein 186–207, 209–257, 264–277, 282–299,301–309, 312–318, C: 36, D: 4 324–339, 358–368, 372–378, 387–397,425–431 gbs0048 Hypothetical protein 29–38, 44–64, 70–76, 78–87, 94–100,102–112, 119–134, 140–149, K: 13 73–92 5, 222 163–173, 178–186, 188–194,207–234, 247–262, 269–290 gbs0053 aldehyde-alcohol 10–28, 36–63, 77–87,103–119, 127–136, 141–169, 171–183, 195–200, E: 4 757–774 6, 223dehydrogenase 207–232, 236–246, 251–265, 268–283, 287–297, 314–322,(adhE) 335–343, 354–363, 384–390, 405–411, 419–436, 443–455, 467–473,480–513, 518–529, 550–557, 565–585, 602–608, 616–625, 632–660, 665–677,685–701, 726–736, 738–747, 752–761, 785–796, 801–813, 838–853, 866–871gbs0061 rplB ribosomal 31–38, 61–66, 74–81, 90–115, 123–145, 154–167,169–179, 182–193, F: 2, I: 12 235–251 7, 224 protein L2 200–206,238–244, 267–272 gbs0084 DNA-directed RNA 19–25, 38–54, 56–64, 66–72,74–92, 94–100, 116–129, 143–149, C: 4, D: 6 241–313 8, 225 polymerase,alpha 156–183, 204–232, 253–266, 269–275, 294–307 subunit (rpoA) gbs0107conserved 5–34, 50–56, 60–65, 74–85, 89–97, 108–119, 159–165, 181–199,K: 2 64–75 9, 226 hypothetical protein 209–225, 230–240, 245–251,257–262, 274–282, 300–305 gbs0108 deoxyuridine 5′- 5–13, 16–21, 27–42,45–52, 58–66, 74–87, 108–114, 119–131 I: 5 39–51 10, 227 triphosphatenucleotidohydrolase gbs0113 ribose ABC 6–23, 46–54, 59–65, 78–84,100–120, 128–133, 140–146, 159–165, F: 4 267–274 11, 228 transporter171–183, 190–204, 224–232, 240–248, 250–259, 274–280, 288–296, 306–315gbs0123 similar to 5–12, 15–24, 26–36, 42–65, 68–80, 82–104, 111–116,125–144, K: 17 162–174 12, 229 argininosuccinate 159–167, 184–189,209–218, 235–243, 254–265, 269–283, synthase 287–300, 306–316, 318–336,338–352, 374–392 gbs0127 rpmV 50S ribosomal 30–42, 45–54 F: 11 25–37 13,230 protein L28 gbs0144 oligopeptide ABC 10–30, 53–59, 86–95, 116–130,132–147, 169–189, 195–201, 212–221, E: 7 419–431 14, 231 transporter,247–256, 258–265, 278–283, 291–298, 310–316, 329–339, substrate-binding341–352, 360–367, 388–396, 398–411, 416–432, 443–452, 460–466, 506–512,515–521, 542–548 gbs0183 membrane protein, 4–27, 30–53, 60–67, 70–90,92–151, 159–185, 189–195, 198–210, F: 9 173–189 15, 232 putative 215–239gbs0184 oligopeptide ABC 4–26, 41–54, 71–78, 116–127, 140–149, 151–158,161–175, 190–196, E: 6 174–188 16, 233 transporter, 201–208, 220–226,240–252, 266–281, 298–305, 308–318, oligopeptide-binding 321–329,344–353, 372–378, 384–405, 418–426, 429–442, 457–463, 494–505, 514–522gbs0235 glycine 17–25, 27–39, 61–67, 81–89, 99–110, 120–131, 133–139,147–161, G: 8, H: 15 239–256 17, 234 betaine/carnitine/choline 167–172,179–185, 192–198, 203–213, 226–238, 243–258, ABC transporter 261–267,284–290, 296–307, 311–328, 340–352, 356–371 gbs0255 conserved 8–30,40–49, 67–80, 114–123, 126–142, 152–162, 188–194 E: 2 57–70 18, 235hypothetical protein gbs0260 glycyl-tRNA 4–23, 28–34, 36–47, 50–61,76–81, 89–94, 96–104, 112–119, 126–146, F: 3 282–297 19, 236 synthetase(beta 155–181, 195–200, 208–214, 220–229, 244–260, 263–276, subunit)282–288, 292–300, 317–323, 336–351, 353–359, 363–375, 382–399, 415–432,444–455, 458–471, 476–481, 484–492, 499–517, 522–529, 535–541, 543–568,572–584, 586–600, 607–617, 626–637, 656–675 gbs0268 transketolase (tkt)6–24, 30–35, 38–45, 63–91, 134–140, 146–160, 167–188, 214–220, E: 787–97 20, 237 226–234, 244–250, 260–270, 286–301, 316–329, 340–371,429–446, 448–459, 474–481, 485–491, 512–526, 537–544, 550–565, 573–583,596–613, 621–630, 652–658 gbs0286 NH3-dependent 8–20, 26–48, 56–67,76–86, 94–109, 115–121, 123–129, 143–160, F: 12, G: 7, H: 8 237–247 21,238 NAD+ synthetase 178–186, 191–198, 201–208, 221–236, 238–244, 260–268gbs0288 similar to penicillin- 4–40, 42–57, 73–87, 98–117, 126–135,150–156, 166–174, 196–271, D: 5, K: 3 448–528 22, 239 binding protein 1A231–236, 248–258, 276–284, 293–301, 307–313, 339–347, 359–365, 375–387,395–402, 428–440, 445–456, 485–490, 497–505, 535–541, 547–555, 610–625,648–656, 665–671 gbs0343 seryl-tRNA 10–18, 39–45, 51–61, 80–96, 98–106,110–115, 158–172, 174–183, I: 3 322–338 23, 240 synthetase (serS)191–200, 220–237, 249–255, 274–289, 308–324, 331–341, 372–381, 384–397,405–414 gbs0411 Hypothetical protein 30–36, 38–56, 85–108, 134–147,149–160, 163–183, 188–201, I: 11 5–13 24, 241 206–211, 219–238, 247–254gbs0428 similar to fibrinogen 11–40, 98–103, 110–115, 133–145, 151–159,172–179, 192–201, A: 7, B: 2, C: 31 1–148 25, 242 binding protein,204–212, 222–228, 235–245, 258–268, 283–296, 298–309, putative 322–329,342–351, 354–362, 372–378, 385–393, 407–418, peptidoglycan linked495–516 protein (LPXTG motif) gbs0437 glucose-6-phosphate 5–19, 21–36,73–94, 112–119, 122–137, 139–145, 152–167, 184–190, I: 26 113–140 26,243 isomerase (pgi) 198–204, 208–224, 249–265, 267–281, 299–304,309–317, 326–333, 356–364, 368–374, 381–389, 391–414, 419–425, 430–435gbs0460 decarboxylase 45–54, 59–67, 78–91 I: 7, K: 11 15–23 27, 244gbs0465 oxydoreductase 11–22, 33–47, 52–80, 88–112, 124–129 F: 4 6–2528, 245 gbs0470 similar to alpha 26–41, 51–63, 80–89, 93–115, 150–163,187–193, 220–237, 240–249, B: 4, C: 2, D: 8 420–511, 581–704 29, 246protein, putative 286–294, 296–306, 316–329, 345–353, 361–370, 407–425,peptidoglycan linked 428–437, 474–482, 484–494, 504–517, 533–541,549–558, protein (LPXTG 595–613, 616–625, 660–668, 673–685, 711–726,736–744, motif) 749–761, 787–802, 812–820, 825–837, 863–878, 888–896,901–913, 939–954, 964–972, 977–989, 1003–1008, 1016–1022, 1028–1034,1041–1053, 1059–1074, 1101–1122 gbs0489 acetyltransferase, 18–25, 27–55,71–83, 89–95, 102–113, 120–146, 150–156, 174–185 E: 32 159–175 30, 247GNAT family gbs0492 gbs0492 valyl-tRNA 24–30, 38–56, 63–68, 87–93,136–142, 153–164, 183–199, 213–219, A: 3 806–884 31, 248 synthetase226–234, 244–261, 269–278, 283–289, 291–297, 320–328, 330–336, 340–346,348–356, 358–366, 382–387, 401–408, 414–419, 449–455, 468–491, 504–512,531–537, 554–560, 597–608, 621–627, 632–643, 650–662, 667–692, 703–716,724–737, 743–758, 783–794, 800–818, 846–856 gbs0538 amino acid ABC 4–14,21–39, 86–92, 99–107, 121–131, 136–144, 147–154, 158–166, G: 1 117–13632, 249 transporter (ATP- 176–185, 193–199, 207–222, 224–230 bindingprotein) gbs0539 similar to 65–76, 85–97, 103–109, 115–121, 125–146,163–169, 196–205, K: 4 266–284 33, 250 phosphomannomutase 212–219,228–237, 241–247, 254–262, 269–288, 294–303, 305–313, 328–367, 395–401,405–412, 418–429, 437–447, 481–488, 506–513, 519–524, 530–541, 546–557gbs0555 beta-lactam 5–14, 37–42, 49–71, 78–92, 97–112, 127–136, 147–154,156–163, E: 3 194–223 34, 251 resistance factor 186–198, 216–225,233–243, 248–253, 295–307, 323–332, (fibA) 359–366, 368–374, 380–398gbs0579 dipeptidase 4–11, 33–39, 45–72, 100–113, 119–129, 136–144,169–175, 177–185, I: 4 438–454 35, 252 200–208, 210–219, 262–276,278–297, 320–326, 336–344, 347–362, 381–394, 443–453 gbs0580 zinc ABCtransporter, 4–29, 31–52, 55–61, 95–110, 138–158, 162–171, 179–187,202–229, I: 11 161–178 36, 253 zinc-binding 239–248, 251–256, 262–267,269–285, 304–310, 351–360, adhesion, lipoprotein 362–368, 381–388,415–428, 435–440, 448–458 gbs0628 cell wall surface 4–17, 19–28, 32–43,47–59, 89–110, 112–126, 128–134, 140–148, I: 9, H: 1 305–381 37, 254anchor family protein 152–161, 169–184, 191–204, 230–235, 255–264,328–338, (IPxTG) 341–347, 401–409, 413–419, 433–441, 449–458, 463–468,476–482, 486–492, 500–506, 529–545 gbs0632 cell wall surface 10–29,38–45, 53–61, 134–145, 152–160, 163–170, 202–208, 219–229, H: 3 698–71538, 255 anchor family 248–258, 266–275, 282–288, 315–320, 328–334,377–385, protein, putative 392–402, 418–424, 447–453, 460–471, 479–487,491–497, (FPKTG motive) 500–507, 531–537, 581–594, 615–623, 629–635,644–652, 659–666, 668–678, 710–717, 719–728, 736–741, 747–760, 766–773,784–789, 794–800, 805–817, 855–861, 866–887 gbs0634 putative surface16–26, 29–37, 44–58, 62–68, 74–80, 88–95, 97–120, 125–144, 165–196 H: 158–72 39, 256 protein gbs0667 regulatory protein, 14–21, 23–46, 49–60,63–74, 78–92, 96–103, 117–129, 134–161, I: 2 243–257 40, 257 putative,truncation 169–211, 217–231, 239–248, 252–281, 292–299, 313–343 gbs0672transcriptional 11–27, 46–52, 67–72, 76–84, 91–112, 116–153, 160–175,187–196, G: 11 43–76 41, 258 regulator (GntR 202–211, 213–220 family)gbs0687 Hypothetical protein 5–29, 37–56, 78–86, 108–118, 152–161 I: 4120–130 42, 259 gbs0785 Similar to penicillin 8–14, 19–41, 52–66, 75–82,87–92, 106–121, 127–133, 136–143, K: 3 184–196 43, 260 binding protein2B 158–175, 180–187, 196–204, 221–228, 239–245, 259–265, 291–306,318–323, 328–340, 352–358, 361–368, 375–381, 391–399, 411–418, 431–442,446–455, 484–496, 498–510, 527–533, 541–549, 558–565, 575–585, 587–594,644–655, 661–668, 671–677 gbs0811 beta-glucosidase 4–22, 29–38, 55–62,75–81, 102–107, 110–134, 143–150, 161–167, L: 13 198–218 44, 261172–179, 191–215, 223–233, 241–247, 251–264, 266–272, 288–309, 340–352,354–366, 394–402, 414–438 gbs0828 hypothetical protein 24–44, 49–70,80–91, 105–118, 128–136, 140–154 I: 3 77–92 45, 262 gbs0851 hypotheticalprotein 5–22, 31–36, 41–47, 67–74, 83–90, 105–122, 135–143, 160–167 E: 2118–129 46, 263 gbs0865 hypothetical protein 4–25, 33–73, 81–93, 96–106,114–120, 122–128, 130–172, 179–208, K: 4 92–100 47, 264 210–241,251–283, 296–301 gbs0890 exonuclease RexB 14–24, 29–38, 43–50, 52–72,86–97, 101–107, 110–125, 127–141, E: 5 336–349 48, 265 (rexB) 145–157,168–175, 177–184, 186–195, 205–226, 238–250, 255–261, 284–290, 293–304,307–314, 316–323, 325–356, 363–371, 383–390, 405–415, 423–432, 442–454,466–485, 502–511, 519–527, 535–556, 558–565, 569–574, 612–634, 641–655,672–686, 698–709, 715–722, 724–732, 743–753, 760–769, 783–792, 818–825,830–839, 842–849, 884–896, 905–918, 926–940, 957–969, 979–1007,1015–1021, 1049–1057 gbs0896 similar to acetoin 6–16, 26–31, 33–39,62–73, 75–85, 87–100, 113–123, 127–152, K: 2 181–195 49, 266dehydrogenase 157–164, 168–181, 191–198, 208–214, 219–226, 233–254,259–266, 286–329 gbs0898 acetoin 4–13, 32–39, 53–76, 99–108, 110–116,124–135, 137–146, 149–157, E: 13, F: 2, I: 2, 31–45, 419–443 50, 267dehydrogenase, 162–174, 182–190, 207–231, 242–253, 255–264, 274–283, J:2 thymine PPi 291–323, 334–345, 351–360, 375–388, 418–425, 456–474,dependent 486–492, 508–517, 520–536, 547–560, 562–577 gbs0904phosphoglucomutase/ 15–26, 30–37, 42–49, 58–90, 93–99, 128–134, 147–154,174–179, I: 3 246–256 51, 268 phosphomannomutase 190–197, 199–205,221–230, 262–274, 277–287, 300–314, family protein 327–333, 343–351,359–377, 388–396, 408–413, 416–425, 431–446 gbs0918 weakly similar to5–26, 34–42, 47–54, 61–67, 71–104, 107–115, 131–138, 144–153, B: 5, C:11, 14–138, 166–286, 52, 269 histidine triad 157–189, 196–202, 204–210,228–245, 288–309, 316–329, D: 36, E: 3, K: 3 372–503, protein, putative332–341, 379–386, 393–399, 404–412, 414–421, 457–468, 674–696, 754–859lipoprotein 483–489, 500–506, 508–517, 523–534, 543–557, 565–580,587–605, 609–617, 619–627, 631–636, 640–646, 662–668, 675–682, 705–710,716–723, 727–732, 750–758, 784–789, 795–809, 869–874 gbs0931 pyruvatekinase 5–17, 32–38, 40–47, 80–89, 113–119, 125–137, 140–154, 157–163, F:78 116–124 53, 270 170–177, 185–199, 213–225, 228–236, 242–248, 277–290,292–305, 323–333, 347–353, 364–370, 385–394, 399–406, 423–433, 441–451,462–474, 477–487 gbs0947 similar to L-Lactate 7–16, 18–30, 32–49, 53–61,63–85, 95–101, 105–115, 119–134, K: 28 63–72 54, 271 Dehydrogenase143–150, 159–178, 185–202, 212–229, 236–250, 254–265, 268–294 gbs0948DNA gyrase, A 4–12, 19–47, 73–81, 97–103, 153–169, 188–198, 207–213,217–223, I: 4 358–370 55, 272 subunit (gyrA) 236–242, 255–265, 270–278,298–305, 309–317, 335–347, 354–363, 373–394, 419–424, 442–465, 486–492,500–507, 542–549, 551–558, 560–572, 580–589, 607–614, 617–623, 647–653,666–676, 694–704, 706–714, 748–754, 765–772, 786–792, 795–806 gbs0969similar to unknown 18–28, 30–38, 40–46, 49–55, 69–78, 82–98, 104–134,147–153, E: 3 290–305 56, 273 plasmid protein 180–190, 196–202, 218–236,244–261, 266–273, 275–286, 290–295, 301–314, 378–387, 390–395, 427–434gbs0971 similar to putative 4–13, 20–31, 39–51, 54–61, 69–84, 87–105,117–124 K: 17 108–125 57, 274 plasmid replication protein gbs0972Hypothetical protein 24–34, 43–54, 56–66, 68–79 E: 3 50–69 58, 275gbs0983 similar to plasmid 5–43, 71–77, 102–131, 141–148, 150–156,159–186, 191–207, D: 11, E: 2, F: 2, 165–178, 818–974 59, 276 protein209–234, 255–268, 280–286, 293–299, 317–323, 350–357, J: 10, K: 10,363–372, 391–397, 406–418, 428–435, 455–465, 484–497, L: 46, M: 3499–505, 525–531, 575–582, 593–607, 621–633, 638–649, 655–673, 684–698,711–725, 736–741, 743–752, 759–769, 781–793, 813–831, 843–853, 894–905,908–916, 929–946, 953–963, 970–978, 1001–1007, 1011–1033 gbs0986 surfaceantigen 16–44, 63–86, 98–108, 185–191, 222–237, 261–274, 282–294, B: 3,C: 12, 77–90, 144–212, 60, 277 proteins, putative 335–345, 349–362,374–384, 409–420, 424–430, 440–447, D: 2, E: 3, F: 20, 279–355,peptidoglycan bound 453–460, 465–473, 475–504, 522–534, 538–551,554–560, H: 3, I: 3, J: 5, 434–536, 782–810, 567–582, 598–607, 611–619,627–640, 643–653, 655–661, M: 2 875–902 669–680, 684–690, 701–707,715–731, 744–750, 756–763, 768–804, 829–837, 845–853, 855–879, 884–890,910–928 gbs0988 similar to plasmid 4–22, 29–41, 45–51, 53–66, 70–77,86–95, 98–104, 106–124, 129–135, J: 2 212–227 61, 278 surface exclusion142–151, 153–161, 169–176, 228–251, 284–299, 331–337, protein, putative339–370, 380–387, 393–398, 406–411, 423–433, 440–452, peptidoglycanbound 461–469, 488–498, 501–516, 523–530, 532–559, 562–567, protein(LPXTG 570–602, 612–628, 630–645, 649–659, 666–672, 677–696, motif)714–723, 727–747 gbs0991 ATP-dependent Clp 4–9, 17–31, 35–41, 56–61,66–75, 81–87, 90–124, 133–138, 149–163, F: 15 275–291 62, 279 protease,ATP- 173–192, 213–219, 221–262, 265–275, 277–282, 292–298, bindingsubunit 301–307, 333–346, 353–363, 371–378, 419–430, 435–448, ClpA456–469, 551–570, 583–599, 603–612 gbs0993 similar to plasmid 28–34,53–58, 72–81, 100–128, 145–154, 159–168, 172–189, 217–225, B: 2, F: 2,J: 4, 654–669, 63, 280 proteins 227–249, 256–263, 299–309, 322–330,361–379, 381–388, K: 2, M: 7 1400–1483 392–401, 404–417, 425–436,440–446, 451–464, 469–487, 502–511, 543–551, 559–564, 595–601, 606–612,615–626, 633–642, 644–650, 664–670, 674–684, 692–701, 715–723, 726–734,749–756, 763–771, 781–787, 810–843, 860–869, 882–889, 907–917, 931–936,941–948, 951–958, 964–971, 976–993, 1039–1049, 1051–1065, 1092–1121,1126–1132, 1145–1151, 1158–1173, 1181–1192, 1194–1208, 1218–1223,1229–1243, 1249–1254, 1265–1279, 1287–1297, 1303–1320, 1334–1341,1343–1358, 1372–1382, 1406–1417, 1419–1425, 1428–1434, 1441–1448,1460–1473, 1494–1504, 1509–1514, 1529–1550 gbs0995 hypothetical protein10–16, 20–25, 58–65, 97–109, 118–132, 134–146, 148–155, 186–195, H: 3,I: 39, J: 3, 205–230, 733–754 64, 281 226–233, 244–262, 275–284,295–310, 317–322, 330–339, M: 3 345–351, 366–375, 392–403, 408–415,423–430, 435–444, 446–457, 467–479, 486–499, 503–510, 525–537, 540–585,602–612, 614–623, 625–634, 639–645, 650–669, 700–707, 717–724, 727–739gbs0997 hypothetical protein 5–22, 37–43, 72–81, 105–113, 128–133,148–160, 188–194, 204–230, D: 2, F: 52 194–213 65, 282 238–245, 251–257gbs0998 hypothetical protein 16–21, 35–41, 56–72, 74–92, 103–109 I: 262–68 66, 283 gbs1001 hypothetical protein 4–15, 17–82, 90–104, 107–159,163–170, 188–221, 234–245, 252–265 G: 8 220–235 67, 284 gbs1015hypothetical protein 16–22, 36–46, 61–75, 92–107, 113–121, 139–145,148–160 K: 17 30–42 68, 285 gbs1035 conserved 4–12, 20–26, 43–49, 55–62,66–78, 121–127, 135–141, 146–161, E: 3 328–346 69, 286 hypotheticalprotein 164–170, 178–189, 196–205, 233–238, 269–279, 288–318, 325–332,381–386, 400–407 gbs1041 hypothetical protein 5–12, 31–49, 57–63, 69–79,89–97, 99–114, 116–127, 134–142, L: 2 58–68 70, 287 147–154, 160–173,185–193, 199–204, 211–222, 229–236, 243–249, 256–274 gbs1066hypothetical protein 10–20, 28–34, 39–53, 68–79, 84–90, 99–106 K: 273–79 71, 288 gbs1087 FbsA 14–37, 45–50, 61–66, 77–82, 93–98, 109–114,125–130, 141–146, A: 7, B: 2, C: 4, 34–307, 312–385 72, 289 157–162,173–178, 189–194, 205–210, 221–226, 237–242, E: 277, G: 523, 253–258,269–274, 285–290, 301–306, 316–332, 349–359, J: 25 371–378, 385–406gbs1103 ABC transporter 4–10, 17–38, 50–85, 93–99, 109–116, 128–185,189–197, 199–210, I: 5 361–376 73, 290 (ATP-binding 223–256, 263–287,289–312, 327–337, 371–386, 389–394, protein) 406–419, 424–432, 438–450,458–463, 475–502, 507–513, 519–526, 535–542, 550–567 gbs1116 xanthinepermease 10–39, 42–93, 100–144, 155–176, 178–224, 230–244, 246–255, I:48 138–155 74, 291 (pbuX) 273–282, 292–301, 308–325, 332–351, 356–361,368–379, 386–393, 400–421 gbs1126 similar to plasmid 5–11, 17–34, 40–45,50–55, 72–80, 101–123, 145–151, 164–172, E: 2, K: 3 994–1003, 75, 292unknown protein 182–187, 189–195, 208–218, 220–241, 243–252, 255–270,1033–1056 325–331, 365–371, 391–398, 402–418, 422–428, 430–435, 443–452,463–469, 476–484, 486–494, 503–509, 529–553, 560–565, 570–590, 608–614,619–627, 654–661, 744–750, 772–780, 784–790, 806–816, 836–853, 876–885,912–918, 926–933, 961–975, 980–987, 996–1006, 1016–1028, 1043–1053,1057–1062 gbs1143 putative 17–45, 64–71, 73–81, 99–109, 186–192,223–238, 262–275, 283–295, C: 3, D: 2, F: 15, 274–350, 443–513 76, 293peptidoglycan linked 336–346, 350–363, 375–385, 410–421, 425–431,441–448, J: 3 protein (LPXTG) 454–463, 468–474, 476–512, 523–537,539–552, 568–583, 599–608, 612–620, 628–641, 644–654, 656–662, 670–681,685–695, 702–708, 716–723, 725–735, 757–764, 769–798, 800–806, 808–816,826–840, 846–854, 856–862, 874–881, 885–902, 907–928 gbs1145 surfaceexclusion 4–22, 29–41, 45–51, 53–61, 70–76, 85–92, 99–104, 111–122,134–140, C: 2 252–262 77, 294 protein Sec10 142–154, 163–174, 224–232,255–265, 273–279, 283–297, 330–335, 337–348, 356–367, 373–385, 391–396,421–431, 442–455, 475–485, 493–505, 526–538, 544–561, 587–599, 605–620,622–651, 662–670, 675–681, 687–692, 697–712, 714–735 gbs1158 Similar to4–12, 15–35, 40–46, 50–59, 67–94, 110–128, 143–169, 182–188, K: 2 74–9078, 295 oxidoreductase 207–215, 218–228, 238–250 gbs1165 cysteine 9–18,42–58, 78–85, 88–95, 97–106, 115–122, 128–134, 140–145, F: 5 12–29 79,296 desulphurase (iscS-1) 154–181, 186–202, 204–223, 261–267, 269–278,284–293, 300–336, 358–368 gbs1195 staphylokinase and 7–34, 46–53, 62–72,82–88, 100–105, 111–117, 132–137, 144–160, B: 3, C: 2, D: 2, 388–405 80,297 streptokinase 166–180, 183–189, 209–221, 231–236, 246–253, 268–282,G: 3, H: 8 286–293, 323–336, 364–372, 378–392, 422–433 gbs1209ATP-dependent 21–27, 34–50, 72–77, 80–95, 164–177, 192–198, 202–220,226–236, C: 3, I: 5 621–739 81, 298 DNA helicase PcrA 239–247, 270–279,285–292, 315–320, 327–334, 348–355, 364–371, 388–397, 453–476, 488–497,534–545, 556–576, 582–588, 601–607, 609–616, 642–662, 674–681, 687–697,709–715, 721–727, 741–755 gbs1214 conserved 4–14, 16–77, 79–109 B: 225–99 82, 299 hypothetical protein gbs1242 CpsG, beta-1,4- 4–9, 17–23,30–37, 44–55, 65–72, 77–93, 102–121, 123–132, 146–153 L: 24 17–29 83,300 galactosyltransferase gbs1260 ABC transporter, 4–18, 25–41, 52–60,83–92, 104–112, 117–123, 149–155, 159–167, I: 17 124–137 84, 301ATP-binding protein 170–192, 201–210, 220–227, 245–250 gbs1270 gbs1270hyaluronate 8–25, 50–55, 89–95, 138–143, 148–153, 159–169, 173–179,223–238, C: 19, D: 5, 1–128, 252–341, 85, 302 lyase 262–268, 288–295,297–308, 325–335, 403–409, 411–417, L: 19 771–793, 432–446, 463–475,492–501, 524–530, 542–548, 561–574, 1043–1058 576–593, 604–609, 612–622,637–654, 665–672, 678–685, 720–725, 731–739, 762–767, 777–783, 820–838,851–865, 901–908, 913–920, 958–970, 1000–1006, 1009–1015, 1020–1026,1043–1052, 1055–1061 gbs1305 hypothetical protein 16–26, 33–46 I: 264–76 86, 303 gbs1306 Laminin binding 4–27, 69–77, 79–101, 117–123,126–142, 155–161, 171–186, 200–206, A: 6, B: 7, 22–108, 153–318, 87, 304protein 213–231, 233–244, 267–273, 313–329, 335–344, 347–370, C: 17, D:72, 391–527, 374–379, 399–408, 422–443, 445–453, 461–468, 476–482, E: 8,F: 91, G: 2, 638–757 518–534, 544–553, 556–567, 578–595, 601–620,626–636, H: 4, I: 26, J: 3, 646–658, 666–681, 715–721, 762–768, 778–785,789–803, K: 14 809–819 gbs1307 Lmb, laminin- 6–21, 32–43, 62–92,104–123, 135–141, 145–152, 199–216, 218–226, A: 2, D: 3 1–72, 127–21188, 305 binding surface 237–247, 260–269, 274–283, 297–303 proteingbs1308 C5a peptidase, 6–26, 50–56, 83–89, 108–114, 123–131, 172–181,194–200, 221–238, B: 4, C: 15, 1–213, 269–592, 89, 306 authenticframeshift 241–247, 251–259, 263–271, 284–292, 304–319, 321–335, D: 70,E: 18, 992–1120 353–358, 384–391, 408–417, 424–430, 442–448, 459–466, F:26, G: 5, 487–500, 514–528, 541–556, 572–578, 595–601, 605–613, H: 4, J:2, K: 40 620–631, 635–648, 660–670, 673–679, 688–693, 702–708, 716–725,730–735, 749–755, 770–777, 805–811, 831–837, 843–851, 854–860, 863–869,895–901, 904–914, 922–929, 933–938, 947–952, 956–963, 1000–1005,1008–1014, 1021–1030, 1097–1103, 1120–1130, 1132–1140 gbs1309hypothetical protein 9–16, 33–39, 47–59, 65–79, 81–95, 103–108, 115–123,138–148, E: 2, F: 4, H: 2, 95–111, 161–189 90, 307 163–171, 176–185,191–196, 205–211, 213–221, 224–256, J: 2 261–276, 294–302, 357–363,384–390 gbs1311 transposase, C- 21–27, 35–45, 70–76, 92–105, 129–143,145–155, 161–166, 170–191, F: 3 1–18 91, 308 terminal part 204–211,214–231, 234–246, 249–255, 259–275 gbs1321 hypothetical protein 21–35,45–53, 56–64, 69–97 F: 7 1–16 92, 309 gbs1352 putative helicase and25–33, 41–47, 61–68, 86–101, 106–114, 116–129, 134–142, 144–156, E: 3,H: 2, M: 4 748–847, 93, 310 methylase 163–176, 181–190, 228–251,255–261, 276–292, 295–305, 1381–1391 334–357, 368–380, 395–410, 424–429,454–460, 469–482, 510–516, 518–527, 531–546, 558–570, 579–606, 628–636,638–645, 651–656, 668–674, 691–698, 717–734, 742–754, 765–770, 792–797,827–835, 847–859, 874–881, 903–909, 926–933, 942–961, 964–977, 989–1004,1010–1028, 1031–1047, 1057–1075, 1081–1095, 1108–1117, 1138–1144,1182–1189, 1193–1206, 1220–1229, 1239–1246, 1257–1267, 1271–1279,1284–1301, 1312–1320, 1329–1335, 1341–1347, 1358–1371, 1399–1404,1417–1426, 1458–1463, 1468–1476, 1478–1485, 1493–1506, 1535–1541,1559–1574, 1583–1590, 1595–1601, 1603–1611, 1622–1628, 1634–1644,1671–1685, 1689–1696, 1715–1720, 1734–1746, 1766–1775, 1801–1806,1838–1844, 1858–1871, 1910–1917, 1948–1955, 1960–1974, 2000–2015,2019–2036, 2041–2063 gbs1356 Putative 5–12, 18–24, 27–53, 56–63, 96–113,119–124, 131–136, 157–163, C: 5, D: 62, I: 22 187–273, 306–441 94, 311peptidoglycan linked 203–209, 215–223, 233–246, 264–273, 311–316,380–389, protein (LPXTG 393–399, 425–433, 445–450, 457–462, 464–470,475–482, motif) - Agglutinin 507–513, 527–535, 542–548, 550–565,591–602, 607–613, receptor 627–642, 644–664, 673–712, 714–732, 739–764,769–782, 812–818, 826–838, 848–854, 860–871, 892–906, 930–938, 940–954,957–973, 990–998, 1002–1021, 1024–1033, 1037–1042, 1050–1060, 1077–1083,1085–1092, 1100–1129, 1144–1161, 1169–1175, 1178–1189, 1192–1198,1201–1207, 1211–1221, 1229–1239, 1250–1270, 1278–1292, 1294–1300,1314–1335, 1344–1352, 1360–1374, 1394–1405, 1407–1414, 1416–1424,1432–1452, 1456–1462, 1474–1497, 1500–1510, 1516–1522, 1534–1542,1550–1559, 1584–1603, 1608–1627 gbs1376 similar to ATP- 70–80, 90–97,118–125, 128–140, 142–148, 154–162, 189–202, K: 4 207–227 95, 312dependent Clp 214–222, 224–232, 254–260, 275–313, 317–332, 355–360,proteinase (ATP- 392–398, 425–432, 448–456, 464–470, 476–482, 491–505,binding subunit), 521–528, 533–546, 560–567, 592–597, 605–614, 618–626,ClpL 637–644, 646–653, 660–666, 677–691 gbs1377 similar to 5–19, 26–34,37–55, 57–66, 69–83, 86–102, 115–134, 138–143, M: 2 256–266 96, 313homocysteine S- 154–172, 178–195, 209–246, 251–257, 290–302, 306–311methyltransferase gbs1386 hydroxy-3- 10–20, 22–28, 35–57, 72–79, 87–103,108–128, 130–144, 158–171, G: 2 353–365 97, 314 methylglutaryl- 190–198,225–242, 274–291, 301–315, 317–324, 374–385 coenzyme A synthase gbs1390hypothetical protein 4–9, 17–30, 34–54, 59–66, 73–94, 118–130, 135–150,158–171, E: 3, K: 4 89–106, 176–193 98, 315 189–198, 219–239, 269–275,283–301 gbs1391 hypothetical protein 14–20, 22–74, 77–86, 89–99,104–109, 126–135, 154–165, 181–195, E: 3 107–118 99, 316 197–212,216–224, 264–275 gbs1403 similar to 5′- 4–18, 21–38, 63–72, 101–109,156–162, 165–179, 183–192, 195–210, A: 3, C: 12, 1–198 100, 317nucleotidase, 212–218, 230–239, 241–256, 278–290, 299–311, 313–322, D:4, J: 2 putative 332–341, 348–366, 386–401, 420–426, 435–450, 455–460,peptidoglycan bound 468–479, 491–498, 510–518, 532–538, 545–552,557–563, protein (LPXTN) 567–573, 586–595, 599–609, 620–626, 628–636,652–657, 665–681 gbs1408 Similar to ABC 4–10, 16–38, 51–68, 73–79,94–115, 120–125, 132–178, 201–208, D: 2, K: 4 191–206 101, 318transporter (ATP- 216–223, 238–266, 269–295, 297–304, 337–342, 347–356,binding protein) 374–401, 403–422, 440–447, 478–504, 510–516, 519–530,537–544 gbs1420 similar to cell wall 12–40, 42–48, 66–71, 77–86, 95–102,113–120, 129–137, 141–148, C: 3, D: 4 370–478 102, 319 proteins,putative 155–174, 208–214, 218–225, 234–240, 256–267, 275–283,peptidoglycan linked 300–306, 313–321, 343–350, 359–367, 370–383,398–405, protein (LPXTG 432–439, 443–461, 492–508, 516–526, 528–535motif) gbs1429 hypothetical protein 6–14, 20–37, 56–62, 90–95, 97–113,118–125, 140–145, 161–170, B: 2, C: 2 176–187 103, 320 183–202, 237–244,275–284, 286–305, 309–316, 333–359, 373–401, 405–412 gbs1442hypothetical 33–44, 50–55, 59–80, 86–101, 129–139, 147–153, 157–163,171–176, L: 28 71–88, 353–372 104, 321 thiamine biosynthesis 189–201,203–224, 239–245, 257–262, 281–287, 290–297, protein, ThiI 304–320,322–331, 334–350, 372–390, 396–401 gbs1452 rplT 50S ribosomal 5–11,15–24, 26–33, 40–47, 75–88, 95–103, 105–112 E: 2 17–30 105, 322 proteinL20 gbs1464 ferrichrome ABC 5–11, 16–39, 46–54, 62–82, 100–107, 111–124,126–150, 154–165, F: 4 8–16 106, 323 transporter 167–183, 204–238,245–295, 301–313, 316–335 (permease) gbs1470 conserved 4–19, 34–48,69–74, 79–107, 115–127, 129–135, 143–153, 160–169, I: 4 142–153 107, 324hypothetical protein 171–182 gbs1528 conserved 4–30, 65–74, 82–106,110–120, 124–132, 135–140, 146–175, 179–184, I: 7 174–186 108, 325hypothetical protein 190–196, 217–223, 228–233, 250–267, 275–292,303–315, 322–332 gbs1529 Putative 9–16, 29–41, 47–57, 68–84, 87–109,113–119, 162–180, 186–193, C: 2 883–936 109, 326 peptidoglycan bound195–201, 203–208, 218–230, 234–243, 265–271, 281–292, protein (LPXTG305–312, 323–332, 341–347, 349–363, 368–374, 383–390, motif) 396–410,434–440, 446–452, 455–464, 466–473, 515–522, 529–542, 565–570, 589–600,602–613, 618–623, 637–644, 1019–1027, 1238–1244, 1258–1264, 1268–1276,1281–1292, 1296–1302 gbs1531 UvrB excinuclease 10–17, 23–32, 39–44,54–72, 75–81, 88–111, 138–154, 160–167, M: 2 384–393 110, 327 ABC chainB 178–185, 201–210, 236–252, 327–334, 336–342, 366–376, 388–400,410–430, 472–482, 493–526, 552–558, 586–592, 598–603, 612–621, 630–635,641–660 gbs1533 glutamine ABC 4–22, 24–39, 50–59, 73–84, 100–105,111–117, 130–138, 155–161, I: 4 445–461 111, 328 transporter, 173–178,182–189, 205–215, 266–284, 308–313, 321–328, glutamine-binding 330–337,346–363, 368–374, 388–395, 397–405, 426–434, protein 453–459, 482–492,501–507, 509–515, 518–523, 527–544, 559–590, 598–612, 614–629, 646–659,663–684, 686–694, 698–721 gbs1536 hypothetical protein 14–22, 27–33 E:10 3–17 112, 329 gbs1542 oxidoreductase, 29–41, 66–73, 81–87, 90–108,140–146, 150–159, 165–184, 186–196, I: 13 126–140 113, 330 aldo/ketoreductase 216–226, 230–238, 247–253, 261–269 family gbs1547 smallprotein, SmpB 5–12, 16–25, 27–33, 36–45, 60–68, 83–88, 103–126 L: 1186–101 114, 331 gbs1565 hypothetical protein 14–23, 36–47, 56–66, 84–89,94–105, 111–127, 140–153, 160–174, F: 2, J: 2, K: 8, 194–227 115, 332176–183, 189–203, 219–225, 231–237, 250–257 L: 21 gbs1586peptidyl-prolyl cis- 4–25, 54–60, 64–71, 73–82, 89–106, 117–124,157–169, 183–188, E: 3 58–98 116, 333 trans isomerase, 199–210, 221–232,236–244, 255–264 cyclophilin-type gbs1591 5- 13–19, 26–36, 41–53, 55–71,77–84, 86–108, 114–135, 157–172, I: 18, L: 2 110–125, 156–170 117, 334methylthioadenosine 177–183, 187–194, 208–213, 218–226 nucleosidase/S-adenosylhomocysteine nucleosidase (pfs) gbs1632 similar to branched-5–24, 63–69, 77–85, 94–112, 120–137, 140–146, 152–159, 166–172, B: 2, E:4, I: 3 1–56, 340–352 118, 335 chain amino acid 179–187, 193–199,206–212, 222–228, 234–240, 244–252, ABC transporter, 257–264, 270–289,298–309, 316–328, 337–348, 363–375 amino acid-binding protein gbs1638amino acid permease 18–39, 42–71, 78–120, 124–144, 152–173, 179–189,199–209, E: 8, G: 9, H: 9 313–327 119, 336 213–222, 228–258, 269–304,329–361, 364–372, 374–389, 396–441 gbs1662 conserved 19–25, 91–98,108–120, 156–162, 168–174, 191–204, 211–216, E: 3, H: 2 10–25, 322–338120, 337 hypothetical protein 232–266, 272–278, 286–308, 316–321,327–333, 344–355, 358–364, 384–391, 395–428, 464–476, 487–495, 497–511,544–561, 563–573, 575–582, 588–594 gbs1666 SWI/SNF family 14–26, 32–49,51–57, 59–72, 80–91, 102–112, 119–125, 147–161, F: 4 90–103 121, 338helicase 164–173, 175–183, 188–213, 217–222, 246–254, 260–276, 282–303,308–318, 321–328, 333–350, 352–359, 371–378, 392–401, 407–414, 416–443,448–463, 471–484, 490–497, 501–514, 519–527, 539–551, 557–570, 578–590,592–598, 600–610, 618–629, 633–647, 654–667, 676–689, 702–709, 718–726,728–737, 741–760, 764–780, 786–795, 808–826, 836–842, 845–852, 865–874,881–887, 931–945, 949–957, 968–974, 979–986, 1003–1009, 1023–1029gbs1673 conserved 11–16, 37–56, 60–66, 69–77, 80–88, 93–106, 117–139,166–171 E: 2 72–90 122, 339 hypothetical protein gbs1695dihydroxyacetone 59–84, 123–133, 145–150, 161–167, 178–189 I: 8 115–128123, 340 kinase family protein gbs1754 excinuclease ABC, A 15–33, 39–46,52–64, 74–87, 108–124, 127–144, 150–156, 173–179, I: 2 132–142 124, 341subunit (uvrA) 184–194, 201–208, 219–236, 243–269, 272–295, 302–309,343–349, 356–361, 370–379, 405–411, 414–423, 430–451, 457–464, 466–475,477–483, 496–502, 507–522, 541–548, 557–563, 571–577, 579–585, 590–605,626–642, 650–662, 671–691, 704–710, 751–769, 775–781, 786–791, 794–829,851–858, 868–878, 884–904, 913–919, 931–939 gbs1760 Similar to A/G-33–58, 64–71, 74–80, 83–88, 96–120, 122–139, 146–157, 167–177, K: 897–115, 199–211 125, 342 specific adenine 207–213, 220–225, 236–242,264–279, 300–305, 326–336, glycosylase 340–347, 350–360 gbs1777 glyceroluptake 4–26, 43–57, 70–99, 102–117, 121–133, 142–148, 151–168, 170–183,E: 4 30–41 126, 343 facilitator protein, 192–220, 235–249, 258–279putative gbs1783 polyprenyl 34–42, 48–58, 70–94, 110–130, 154–160,164–172, 178–183, 195–203, I: 3 222–233 127, 344 synthetase family211–222, 229–250, 256–261, 274–284, 286–292, 312–323 protein gbs1784 ABCtransporter, 4–9, 15–36, 38–45, 49–74, 78–88, 100–112, 136–191, 211–220,C: 2, D: 2 208–280 128, 345 ATP-binding protein 226–233, 239–246,254–274, 287–307, 316–322, 342–353, CydC 356–366, 373–378, 384–393,405–431, 449–457, 459–468, 487–511, 515–524, 529–541, 544–552, 562–568,571–576 gbs1790 hypothetical protein 10–27, 31–37, 39–54, 71–108,124–143 A: 23, C: 6 2–107 129, 346 gbs1805 Similar to secreted 16–27,38–57, 64–70, 90–102, 104–113, 116–137, 160–166 A: 197, C: 2 1–80 130,347 unknown protein gbs1816 HD domain protein 13–21, 31–36, 56–67,127–136, 153–171, 173–180, 184–200, 214–222, F: 8 135–159 131, 348225–231, 239–263, 267–273 gbs1821 Similar to 23S 12–27, 31–51, 68–74,77–87, 94–101, 108–114, 117–123, 127–134, K: 5 205–223 132, 349ribosomal RNA 138–168, 173–196, 201–207, 212–217, 227–237, 247–257,methyltransferase 264–280 gbs1823 triad family protein 17–22, 25–54,70–76, 92–100 G: 6, H: 3 98–110 133, 350 gbs1834 two-component 7–29,40–50, 60–67, 87–96, 105–111, 119–164, 172–199, 206–212, E: 3, F: 6159–176 134, 351 sensor histidine 220–227, 237–259, 272–279, 282–293,295–309, 313–319, kinase 321–328, 345–363, 376–386 gbs1842transcriptional 4–19, 24–30, 36–43, 50–68, 71–89, 93–106, 141–152,154–172, I: 19 362–377 135, 352 antiterminator, BglG 179–197, 199–215,229–239, 246–252, 255–263, 281–298, family 319–325, 329–356, 358–368,374–390, 397–409, 420–429, 432–444, 450–456, 459–475, 483–494, 496–502,520–528, 532–556 gbs1850 hypothetical 18–25, 40–62, 77–85, 91–97,105–116, 123–133, 139–184, 189–197 G: 2 122–140 136, 353 transaldolasegbs1869 phosphoglycerate 4–49, 52–58, 62–70, 79–105, 109–133, 142–150,163–168, 206–214, L: 9 643–658 137, 354 kinase 220–228, 233–240,243–254, 274–281, 303–311, 327–338, 357–373, 378–396, 403–413, 420–436,441–453, 461–467, 475–481, 484–498, 506–512, 514–521, 523–529, 562–579,589–595, 598–603, 615–648, 714–722, 728–742, 749–758, 777–792, 795–807gbs1875 alkyl hydroperoxide 8–27, 37–48, 51–56, 72–79, 87–106, 120–138,140–147, 167–176, F: 3 456–470 138, 355 reductase (large 187–197,205–216, 222–229, 234–239, 243–249, 277–288, subunit) and NADH 292–315,334–343, 347–353, 363–391, 398–404, 430–447, dehydrogenase 461–467,478–492, 498–507 gbs1879 endopeptidase O 5–12, 18–24, 59–69, 80–93,95–109, 119–125, 130–137, 139–147, I: 26 221–237 139, 356 (pepO)158–163, 168–176, 182–202, 206–215, 222–239, 241–249, 267–277, 291–298,311–318, 321–327, 338–344, 348–355, 373–386, 393–406, 411–417, 434–443,446–465, 473–484, 514–521, 532–553, 584–594 gbs1893 2-keto-3- 4–14,27–34, 50–58, 63–72, 79–106, 109–114, 121–142, 146–154, F: 8, K: 9167–191 140, 357 deoxygluconate 161–167, 169–175, 178–201, 223–238,249–254, 259–264, kinase 278–292, 294–312, 319–330 gbs1899N-acetylmuramoyl- 7–28, 36–42, 50–61, 63–80, 122–152, 161–174, 176–191B: 2, C: 2, E: 3 140–190 141, 358 L-alanine amidase, family 4 proteingbs1907 citrate carrier protein, 20–57, 59–65, 70–78, 86–102, 119–133,142–161, 163–173, 177–188, I: 2 381–395 142, 359 CCS family 192–202,204–220, 222–236, 240–253, 279–319, 326–331, 337–383, 390–399, 406–412,420–427, 431–438 gbs1924 similar to 13–18, 28–34, 37–43, 50–59, 75–81,83–97, 105–121, 139–147, K: 3 182–201 143, 360 pneumococcal 200–206,209–227, 231–247, 260–271, 318–327, 366–381, histidine triad 388–394,399–406 protein B precursor (C-terminal part) gbs1925 similar to 6–29,37–43, 51–56, 70–77, 82–102, 110–119, 127–143, 178–190, A: 2, B: 5,21–314 144, 361 pneumococcal 201–209, 216–243, 261–269, 281–292,305–313, 327–339, C: 12, D: 57 histidine triad 341–354, 356–373,391–397, 423–429, 438–445, 450–478 protein B precursor (N-terminal part)gbs1962 conserved 4–12, 15–21, 32–41, 59–76, 80–89, 96–104 E: 3 90–103145, 362 hypothetical protein gbs2008 similar to C5A 9–28, 30–41, 44–54,69–74, 77–82, 90–97, 104–123, 125–135, 149–155, A: 253, B: 2, 3–82,509–576 146, 363 peptidase, putative 164–173, 177–184, 217–226, 230–235,238–244, 258–272, C: 3, D: 6, H: 2 peptidoglycan linked 282–297,300–305, 309–315, 317–322, 327–336, 348–362, protein (LPXTG 368–374,380–387, 400–411, 414–424, 451–458, 460–466, motif) 483–494, 497–503,506–511, 521–528, 540–553, 569–587, 598–606, 628–642, 661–681, 688–700,718–733, 740–749, 752–764, 769–783, 823–834, 848–854, 862–872, 878–884,886–898, 915–920, 938–951, 954–961, 963–972, 982–989, 996–1003,1010–1016, 1021–1032, 1038–1044, 1047–1057, 1060–1070, 1079–1088,1094–1102, 1117–1127, 1129–1135, 1142–1153, 1158–1204, 1212–1229,1234–1263, 1269–1277, 1308–1313, 1327–1338, 1344–1376, 1400–1415,1436–1443, 1448–1458, 1497–1504, 1511–1522, 1544–1566 gbs2018 putative8–36, 40–64, 71–79, 88–94, 102–109, 118–127, 138–148, 151–159, A: 132,B: 6, 1–60, 55–139, 147, 364 peptidoglycan linked 163–174, 192–198,200–206, 220–233, 268–273, 290–301, C: 13, D63, 212–308, 386–458,protein (LPXTG 304–309, 316–323, 331–349, 378–391, 414–420, 427–437, E:15, H: 2, J: 9, 458–624 motif) 455–475, 494–510, 541–547, 549–555,616–640 K: 13 gbs2029 hypothetical protein 16–31, 35–42, 70–77, 91–101,120–130, 132–140, 143–153, 185–190, G: 8 273–281 148, 365 195–202,215–222, 228–238, 241–251, 257–264, 268–277, 288–302, 312–324, 326–333,341–348, 364–382, 415–429, 438–454, 458–466, 491–499, 501–521 gbs2042hypothetical protein 8–14, 32–57, 74–149, 155–177, 179–212, 221–266,271–296, 304–324, L: 11 466–490 149, 366 329–346, 349–359, 368–401,413–419, 426–454, 465–478, 493–510 gbs2054 DNA mismatch 22–28, 33–51,64–89, 96–119, 126–132, 138–146, 152–159, 161–169, E: 8 102–113 150, 367repair protein HexA 172–179, 193–198, 205–211, 221–231, 235–254,273–280, 297–303, 312–320, 328–346, 351–373, 378–384, 391–398, 448–454,460–468, 470–481, 516–558, 574–593, 597–602, 613–623, 626–646, 649–656,668–673, 675–683, 696–708, 715–722, 724–739, 745–751, 759–777, 780–804,816–822 gbs2058 hypothetical protein 12–28, 41–91, 98–107, 112–120,125–131, 151–193, 215–221, L: 3 128–138 151, 368 240–250, 263–280gbs2060 aspartyl-tRNA 16–24, 32–38, 46–62, 68–81, 90–105, 127–133,144–150, 160–166, I: 3, L: 12 96–109, 127–139 152, 369 synthetase (aspS)178–184, 186–202, 210–219, 232–240, 252–258, 264–273, 293–324, 337–344,349–357, 360–369, 385–398, 410–416, 419–427, 441–449, 458–476, 508–515,523–539, 544–549, 562–569, 571–579 gbs2075 hypothetical protein 19–25,28–34, 56–61, 85–97, 110–116 M: 2 39–53 153, 370 gbs2106 protein ofunknown 4–37, 41–50, 62–72, 91–97, 99–109, 114–125, 136–141, 149–158, A:5, B: 6, C: 4, 27–225 154, 371 function/lipoprotein, 160–166, 201–215 D:14, E: 11, putative I: 8, K: 23 gbs2118 similar to inosine 15–31, 44–51,96–105, 122–130, 149–157, 162–168, 178–183, K: 17 463–481 155, 372monophosphate 185–192, 198–204, 206–213, 221–234, 239–245, 248–255,dehydrogenase 257–266, 289–335, 349–357, 415–422, 425–441, 448–454,462–468 gbs2131 ABC transporter, 5–31, 39–55, 63–72, 76–99, 106–155,160–177, 179–199, 207–217, I: 2 505–525 156, 373 permease protein,223–240, 245–255, 261–267, 294–316, 321–343, 354–378, putative 382–452,477–488, 529–536, 555–569, 584–591, 593–612, 620–627, 632–640, 647–654,671–680, 698–704, 723–730, 732–750, 769–775, 781–788, 822–852 ARF0112Hypothetical protein none F: 6 3–18 157, 374 ARF0147 Hypotheticalprotein 4–14 E: 3, I: 3 12–24 158, 375 ARF0532 Hypothetical protein4–11, 22–30 F: 10 12–25 159, 376 ARF0534 Hypothetical protein 5–12 E: 2,G: 2 4–18 160, 377 ARF0557 Hypothetical protein 4–28 E: 2, G: 6, H: 47–14 161, 378 ARF0862 Hypothetical protein 6–16 G: 7, H: 4 8–16 162, 379ARF0891 Hypothetical protein 4–15, 18–33 K: 6 24–36 163, 380 ARF0895Hypothetical protein 4–10, 16–21 I: 21 20–31 164, 381 ARF0943Hypothetical protein none C: 2, K: 9 6–19 165, 382 ARF0973 Hypotheticalprotein 11–18 D: 2, G: 3, H: 8, 3–10 166, 383 I: 2, K: 2 ARF0999Hypothetical protein 13–24 B: 4, K: 3 3–15 167, 384 ARF1010 Hypotheticalprotein 15–27 K: 2 7–16 168, 385 ARF1230 Hypothetical protein 11–16 K:11 1–15 169, 386 ARF1503 Hypothetical protein 4–16 E: 13 9–21 170, 387ARF1556 Hypothetical protein 4–24, 40–48, 54–67 F: 2 22–39 171, 388ARF1585 Hypothetical protein 6–30, 34–55, 62–68, 78–106 I: 5, J: 4 68–74172, 389 ARF1588 Hypothetical protein none I: 2 3–14 173, 390 ARF1735Hypothetical protein 9–19 I: 13 6–21 174, 391 ARF1809 Hypotheticalprotein 4–17 H: 2, L: 17 1–9 175, 392 ARF1826 Hypothetical protein 5–30I: 6 1–8 176, 393 ARF1882 Hypothetical protein 4–16, 23–46, 51–56 K: 2345–55 177, 394 ARF1996 Hypothetical protein none F: 3 7–16 178, 395CRF0123 Hypothetical protein none F: 32 2–14 179, 396 CRF0180Hypothetical protein 4–36, 43–65 E: 6, G: 6, H: 12 50–62 180, 397CRF0208 Hypothetical protein 10–30 I: 2 14–21 181, 398 CRF0258Hypothetical protein 9–17 I: 2 1–10 182, 399 CRF0285 Hypotheticalprotein 4–12 F: 2 3–16 183, 400 CRF0311 Hypothetical protein 4–15 H: 45–23 184, 401 CRF0446 Hypothetical protein none L: 20 10–21 185, 402CRF0455 Hypothetical protein none F: 5 6–16 186, 403 CRF0491Hypothetical protein 4–29, 31–38 G: 4 2–14 187, 404 CRF0520 Hypotheticalprotein 4–35 H: 4 33–42 188, 405 CRF0530 Hypothetical protein none G:13, H: 8, K: 3 2–17 189, 406 CRF0570 Hypothetical protein 9–18, 30–35 I:2 15–33 190, 407 CRF0649 Hypothetical protein 4–9 G: 8, H: 6 6–12 191,408 CRF0853 Hypothetical protein none I: 6 3–17 192, 409 CRF0955Hypothetical protein 12–21, 37–44, 52–61, 72–80 E: 7, L: 44 38–48 193,410 CRF0983.1 Hypothetical protein 4–10, 29–44, 54–61, 69–78 K: 59 13–27194, 411 CRF0983.2 Hypothetical protein 13–23, 36–53 L: 33 2–15 195, 412CRF1083 Hypothetical protein 4–25, 28–46, 56–72, 81–99, 120–132,134–142, 154–160 F: 18 129–141 196, 413 CRF1095 Hypothetical protein4–15, 24–33, 35–41, 64–86 L: 15 21–33 197, 414 CRF1212.1 Hypotheticalprotein 9–15 I: 5 4–13 198, 415 CRF1212.2 Hypothetical protein 4–11,13–19, 34–48 L: 30 15–32 199, 416 CRF1290 Hypothetical protein 4–21 I: 711–31 200, 417 CRF1383 Hypothetical protein 23–57 K: 13 38–50 201, 418CRF1416 Hypothetical protein 4–32 E: 16, J: 7 3–13 202, 419 CRF1500Hypothetical protein 4–10, 13–25, 32–42, 56–68, 72–84 E: 16 26–38 203,420 CRF1513 Hypothetical protein 4–20, 31–48, 52–58, 65–71, 80–93,99–108, 114–123 I: 2 37–49 204, 421 CRF1518 Hypothetical protein 6–12,14–20 F: 28 3–25 205, 422 CRF1663 Hypothetical protein 14–25, 27–38 F:10 5–14 206, 423 CRF1667 Hypothetical protein 4–41, 57–105, 109–118,123–136, 144–152 G: 4 86–99 207, 424 CRF1832 Hypothetical protein NoneE: 5, L: 8 6–19 208, 425 CRF1866 Hypothetical protein none G: 3, H: 182–19 209, 426 CRF1892 Hypothetical protein 14–47 L: 11 1–14 210, 427CRF1942 Hypothetical protein 4–21, 29–44 F: 14 2–18 211, 428 CRF1992Hypothetical protein 23–29 K: 10 10–28 212, 429 CRF2047 Hypotheticalprotein 6–16, 22–36 K: 9 11–22 213, 430 CRF2050 Hypothetical protein4–19, 30–44 I: 2 18–27 214, 431 CRF2096 Hypothetical protein 5–15,37–45, 58–65 G: 2 38–47 215, 432 CRF2113 Hypothetical protein 4–15,23–34 I: 5 4–15 216, 433 NRF1311 transposase, C- 30–36, 44–54, 79–85,101–114, 138–152, 154–164, 170–175, 179–200, F: 3 10–28 217, 434terminal part 213–220, 223–240, 243–255, 258–264, 268–284

TABLE 1B Immunogenic proteins identified by amino acid sequence identitywith peptides identified by bacterial surface display. S. agalactiaeantigenic Protein protein Identical SEQ ID Peptide identifiedImmunogenic (new) region Peptide sequence NO name by BSD region (aa)Sequence ID (DNA, protein) gbs0384 210–226 MEYKGNFSQKTINRFKS 489gbs0995.1 gbs0995 210–226 435, 449 738–753 QTQRSGKINTDFMRQL 490gbs0995.2 gbs0995 738–753 gbs0393 326–344 VKTIGYGKLTGKVNHHYVA 491gbs0986.2 gbs0986 326–344 436, 450 326–348 VKTIGYGKLTGKVNHHYVANKDG 492gbs1143.1 gbs1143 327–349 338–354 VNHHYVANKDGSVTAFV 493 gbs0986.3gbs0986 338–354 371–392 AAVNQNIVFRVLTKDGRPIFEK 494 gbs1143.2 gbs1143372–393 801–809 TVIKKGTNL 495 gbs0986.4 gbs0986 801–809 877–901VTHTTEKSKPVEPQKATPKAPAKGL 496 gbs0986.5 gbs0986 877–901 gbs0396 893–906RQELLTPTQLSKLQ 497 gbs0983.1 gbs0983 893–906 437, 451 gbs0407 51–69VRYDKLEALVAYHGAKSAS 498 gbs0972.1 gbs0972 51–69 438, 452 gbs0408 110–125HQPNRIYLTDKLVPYI 499 gbs0971.1 gbs0971 110–125 439, 453 gbs0410 291–305QSIKQHDKEKLRTVL 500 gbs0969.1 gbs0969 291–305 440, 454 gbs0714 210–226MEYKGNFSQKTINRFKS 501 gbs0995.1 gbs0995 210–226 441, 455 738–753QTQRSGKINTDFMRQL 502 gbs0995.2 gbs0995 738–753 gbs0723 326–344VKTIGYGKLTGKVNHHYVA 503 gbs0986.2 gbs0986 326–344 442, 456 326–348VKTIGYGKLTGKVNHHYVANKDG 504 gbs1143.1 gbs1143 327–349 338–354VNHHYVANKDGSVTAFV 506 gbs0986.3 gbs0986 338–354 371–392AAVNQNIVFRVLTKDGRPIFEK 506 gbs1143.2 gbs1143 372–393 801–809 TVIKKGTNL507 gbs0986.4 gbs0986 801–809 877–901 VTHTTEKSKPVEPQKATPKAPAKGL 508gbs0986.5 gbs0986 877–901 gbs0726 893–906 RQELLTPTQLSKLQ 509 gbs0983.1gbs0983 893–906 443, 457 gbs0737 51–69 VRYDKLEALVAYHGAKSAS 510 gbs0972.1gbs0972 51–69 444, 458 gbs0738 110–125 HQPNRIYLTDKLVPYI 511 gbs0971.1gbs0971 110–125 445, 459 gbs0740 291–305 QSIKQHDKEKLRTVL 512 gbs0969.1gbs0969 291–305 446, 460 gbs0897 32–44 EGDVLLEIMSDKT 513 gbs0898.1gbs0898 32–44 447, 461 gbs0966 399–410 PGLTVEEKFVTF 514 gbs0144.1gbs0144 420–431 448, 462

TABLE 2 Epitope serology with human sera Peptides positivity aa from aato Seq ID gbs0012.1 ++ 120 143 218 gbs0012.2 + 138 161 218 gbs0012.3 +156 179 218 bbs0016.2 +++ 110 129 219 gbs0016.3 + 168 184 219gbs0048.1 + 74 90 222 gbs0053.1 +++ 759 773 223 gbs0061.1 +++ 237 260224 gbs0084.1 + 265 284 225 gbs0107.1 ++ 65 74 226 gbs0108.1 ++ 41 50227 gbs0123.1 + 163 174 229 gbs0127.1 ++ 26 37 230 gbs0183.1 + 174 189232 gbs0235.1 ++ 240 256 234 gbs0260.1 + 285 297 236 gbs0286.1 + 238 247238 gbs0288.1 + 491 519 239 gbs0437.1 ++ 114 140 243 gbs0539.1 + 267 284250 gbs0579.1 + 439 453 252 gbs0580.1 ++ 162 178 253 gbs0628.1 ++ 347364 254 gbs0632.1 +++ 699 715 255 gbs0634.1 + 60 71 256 gbs0667.1 ++ 244257 257 gbs0672.1 + 44 63 258 gbs0672.2 + 57 76 258 gbs0785.1 + 185 196260 gbs0851.1 + 119 129 263 gbs0896.1 ++ 182 195 266 gbs0898.1 ++ 32 44267 gbs0898.2 + 424 442 267 gbs0904.1 + 247 256 268 gbs0918.1 ++ 678 694269 gbs0918.2 + 785 805 269 gbs0918.4 + 55 77 269 gbs0918.5 +++ 72 94269 gbs0995.1 + 210 226 281 gbs1087.3 + 37 59 289 gbs1165.1 + 13 29 296gbs1816.1 + 136 159 348 gbs1821.1 + 205 222 349 gbs1823.1 + 99 110 350gbs1834.1 + 160 176 351 gbs1875.1 + 457 470 355 gbs1879.1 + 221 237 356gbs1893.1 + 167 190 357 gbs1925.1 + 96 120 361 gbs2018.3 +++ 399 417 364gbs2018.4 +++ 503 519 364 gbs2018.5 +++ 544 563 364 gbs2106.2 + 46 68371 gbs2106.7 + 159 183 371 gbs2106.8 + 184 198 371 gbs2118.1 ++ 463 481372

TABLE 3 Gene distribution in S. agalactiae strains. Amino acid Seq. Genedistribution substitutions (in ID (DNA, ORF Common name (present of 50)serotype IA strain)* Prot.) gbs0012 weakly similar to beta-lactamase44/44 n.d.  1, 218 gbs0016 glucan-binding protein B (S. mutans) 40/440/224  2, 219 gbs0024 Phosphoribosylformylglycinamidine 46/46 10/228  3, 220 gbs0031 surface immunogenic protein 46/46 1/225  4, 221 gbs0048Unknown 30/46 0/61   5, 222 gbs0053 aldehyde-alcohol dehydrogenase(adhE) 45/45 0/224  6, 223 gbs0061 rplB ribosomal protein L2 46/46 0/218 7, 224 gbs0084 DNA-directed RNA polymerase, alpha 45/45 0/207  8, 225subunit (rpoA) gbs0107 conserved hypothetical protein 46/46 0/235  9,226 gbs0108 deoxyuridine 5′-triphosphate 44/44 0/125 10, 227nucleotidohydrolase gbs0113 ribose ABC transporter 44/45 0/227 11, 228gbs0123 similar to argininosuccinate synthase 44/44 0/184 12, 229gbs0127 rpmV 50S ribosomal protein L28 46/46 0/40  13, 230 gbs0144oligopeptide ABC transporter, substrate- 45/45 0/282 14, 231 bindinggbs0183 membrane protein, putative 44/44 0/223 15, 232 gbs0184oligopeptide ABC transporter, 46/46 1/203 16, 233 oligopeptide-bindinggbs0235 glycine betaine/carnitine/choline ABC 46/46 0/219 17, 234transporter gbs0255 conserved hypothetical protein 46/46 0/180 18, 235gbs0260 glycyl-tRNA synthetase (beta subunit 46/46 0/209 19, 236 gbs0268transketolase (tkt) 46/46 0/208 20, 237 gbs0286 NH3-dependent NAD+synthetase 45/45 0/191 21, 238 gbs0288 similar to penicillin-bindingprotein 1A 45/45 0/212 22, 239 gbs0343 seryl-tRNA synthetase (serS)46/46 0/228 23, 240 gbs0428 similar to fibrinogen binding protein, 45/461/126 25, 242 putative peptidoglycan linked protein (LPXTG motif)gbs0437 glucose-6-phosphate isomerase (pgi) 45/45 0/232 26, 243 gbs0460decarboxylase 46/46 1/81  27, 244 gbs0465 oxydoreductase 46/46 0/126 28,245 gbs0489 acetyltransferase, GNAT family 45/45 3/144 30, 247 gbs0492gbs0492 valyl-tRNA synthetase 44/44 3/125 31, 248 gbs0538 amino acid ABCtransporter (ATP-binding 46/46 0/214 32, 249 protein) gbs0539 similar tophosphomannomutase 46/46 0/244 33, 250 gbs0555 beta-lactam resistancefactor (fibA) 46/46 0/218 34, 251 gbs0579 dipeptidase 46/46 0/218 35,252 gbs0580 zinc ABC transporter, zinc-binding 45/45 2/235 36, 253adhesion liprot gbs0628 cell wall surface anchor family protein - 42/440/219 37, 254 (IPxTG) gbs0632 cell wall surface anchor family protein,44/45 0/238 38, 255 putative (FPKTG motive) gbs0667 regulatory protein,putative, truncation 44/44 0/229 40, 257 gbs0672 transcriptionalregulator (GntR family) 43/43 0/203 41, 258 gbs0687 unknown proteins45/45 0/149 42, 259 gbs0785 Similar to penicillin binding protein 2B45/45 0/218 43, 260 gbs0828 unknown proteins 46/46 1/120 45, 262 gbs0851hypothetical protein 46/46 0/140 46, 263 gbs0865 gbs0865 Unknown 44/440/241 47, 264 gbs0890 exonuclease RexB (rexB) 46/46 0/232 48, 265gbs0896 similar to acetoin dehydrogenase 46/46 0/239 49, 266 gbs0898acetoin dehydrogenase, thymine PPi 45/45 0/180 50, 267 dependent gbs0904phosphoglucomutase/phosphomannomutase 46/46 0/169 51, 268 family protegbs0918 weakly similar to histidine triad protein, 45/45 1/209 52, 269putative lipoprotein gbs0931 pyruvate kinase 46/46 0/185 53, 270 gbs0947similar to L-Lactate Dehydrogenase 46/46 0/233 54, 271 gbs0948 DNAgyrase, A subunit (gyrA) 44/44 0/172 55, 272 gbs1035 conservedhypothetical protein 46/46 0/210 69, 286 gbs1066 gbs1066 Unknown 17/462/92  71, 288 gbs1087 highly repetitive peptidoglycan bound 42/45 n.d.72, 289 protein (LPXTG motif) gbs1103 ABC transporter (ATP-bindingprotein) 46/46 1/165 73, 290 gbs1116 xanthine permease (pbuX) 45/451/170 74, 291 gbs1158 Similar to oxidoreductase 44/44 1/170 78, 295gbs1165 cysteine desulphurase (iscS-1) 43/43  0/148# 79, 296 gbs1195staphylokinase and streptokinase 45/45 60/142 80, 297 gbs1209ATP-dependent DNA helicase PcrA 43/44 1/94# 81, 298 gbs1214 conservedhypothetical protein 43/46 0/97  82, 299 gbs1260 ABC transporter,ATP-binding protein 44/46 1/198 84, 301 gbs1306 Laminin binding protein(Spellerberg, B et 45/46 0/215 87, 304 al 1999) gbs1307 lmblaminin-binding surface protein 45/45 n.d. 88, 305 gbs1308 C5apeptidase, authentic frameshift 46/46 0/205 89, 306 gbs1309 hypotheticalprotein 44/46 0/214 90, 307 gbs1356 Putative peptidoglycan linkedprotein 20/46 50/211# 94, 311 (LPXTG motif) - Agglutinin receptorgbs1376 similar to ATP-dependent Clp proteinase 45/45 0/197 95, 312(ATP-binding subunit), ClpL gbs1377 similar to homocysteine S- 45/450/55  96, 313 methyltransferase gbs1386-hydroxy-3-methylglutaryl-coenzyme A 44/44 0/219 97, 314 synthasegbs1390 gbs1390 Unknown 43/43 0/198 98, 315 gbs1391 gbs1391 Unknown44/44 0/214 99, 316 gbs1403 similar to 5′-nucleotidase, putative 45/453/189 100, 317  peptidoglycan bound protein (LPXTN) gbs1408 Similar toABC transporter (ATP-binding 45/45 0/205 101, 318  protein) gbs1429unknown proteins 46/46 1/193 103, 320  gbs1452 rplT 50S ribosomalprotein L20 46/46 0/101 105, 322  gbs1464 ferrichrome ABC transporter(permease 44/44 2/232 106, 323  gbs1470 conserved hypothetical protein46/46 2/164 107, 324  gbs1528 conserved hypothetical protein 45/45 0/213108, 325  gbs1531 UvrB excinuclease ABC chain B 45/45 0/108 110, 327 gbs1533 glutamine ABC transporter, glutamine- 44/44 0/166 111, 328 binding prote gbs1542 oxidoreductase, aldo/keto reductase family 45/451/219 113, 330  gbs1565 hypothetical protein 43/43 1/218 115, 332 gbs1586 peptidyl-prolyl cis-trans isomerase, 45/45 1/227 116, 333 cyclophilin-type gbs1591 5-methylthioadenosine nucleosidase/S- 45/450/203 117, 334  adenosylhomoc gbs1632 similar to branched-chain aminoacid ABC 45/45 0/223 118, 335  transporter, amino acid-binding proteingbs1638 amino acid permease 45/45 0/100 119, 336  gbs1662 conservedhypothetical protein 45/45 0/213 120, 337  gbs1666 SWI/SNF familyhelicase 45/45 0/200 121, 338  gbs1673 conserved hypothetical protein45/45 0/147 122, 339  gbs1695 dihydroxyacetone kinase family protein43/43 1/165 123, 340  gbs1754 excinuclease ABC, A subunit (uvrA) 43/430/224 124, 341  gbs1760 Similar to A/G-specific adenine glycosylase46/46 0/181 125, 342  gbs1777 glycerol uptake facilitator protein,putative 43/43 0/199 126, 343  gbs1783 polyprenyl synthetase familyprotein 45/45 0/217 127, 344  gbs1784 ABC transporter, ATP-bindingprotein 45/45 1/220 128, 345  CydC gbs1790 unknown proteins 41/43 3/75#129, 346  gbs1805 Similar to secreted unknown proteins 45/45 0/66  130,347  gbs1816 HD domain protein 43/43 1/176 131, 348  gbs1821 Similar to23S ribosomal RNA 43/43  2/155# 132, 349  methyltransferase gbs1834two-component sensor histidine kinase 44/44 0/213 134, 351  gbs1842transcriptional antiterminator, BglG family 43/43 0/208 135, 352 gbs1850 hypothetical transaldolase 44/44 0/194 136, 353  gbs1875 alkylhydroperoxide reductase (large 46/46 0/192 138, 355  subunit) and NADHdehydrogenase gbs1879 endopeptidase O (pepO) 43/43 0/135 139, 356 gbs1893 2-keto-3-deoxygluconate kinase 36/46 0/228 140, 357  gbs1899N-acetylmuramoyl-L-alanine amidase, 43/43 0/149 141, 358  family 4 protegbs1907 citrate carrier protein, CCS family 43/43 0/219 142, 359 gbs1925 similar to pneumococcal histidine triad 43/43 0/103 144, 361 protein B precursor (N-terminal part) gbs1962 conserved hypotheticalprotein 28/46 0/136 145, 362  gbs2008 similar to C5A peptidase, putative43/43 n.d. 146, 363  peptidoglycan linked protein (LPXTG motif) gbs2018putative peptidoglycan linked protein 43/45 0/104 147, 364  (LPXTGmotif) gbs2029 unknown proteins 44/44 1/238 148, 365  gbs2054 DNAmismatch repair protein HexA 46/46 0/206 150, 367  gbs2060 aspartyl-tRNAsynthetase (aspS) 46/46 2/211 152, 369  gbs2106 protein of unknownfunction/lipoprotein, 44/44 0/160 154, 371  putative gbs2118 similar toinosine monophosphate 43/43 0/113 155, 372  dehydrogenase gbs2131 ABCtransporter, permease protein, 45/45 0/237 156, 373  putative

TABLE 4 Immunogenicity of epitopes in mice Peptide ORF aa from aa to IBELISA Seq ID gbs0016 110 129 + 219 168 184 + gbs0986 877 901 + 277 333354 + 326 344 + 801 809 + gbs1805 1 54 +++++ 347 gbs2018 544 563 +++++++ 364 31 51 + +++ 107 119 + 399 417 ++ ++++ 503 519 + ++++ gbs0012120 198 ++ + 218 gbs0016 20 35 + ++ 219 gbs0031 118 201 ++++ 221 gbs042848 132 + +++++ 242 gbs0538 118 136 + ++++ 249 gbs0580 162 178 + + 253gbs0628 347 364 + +++++ 254 gbs0632 699 715 + +++++ 255 gbs0672 50 76 +258 gbs0918 785 819 + +++++ 269 44 128 ++ gbs0971 90 128 +++++ 274gbs1087 314 384 + 289 gbs1143 327 349 +++ 293 gbs1306 242 314 ++ ++++304 405 478 ++ 23 100 + gbs1307 129 210 ++ 305 gbs1309 162 188 ++ 307gbs1352 750 772 ++ +++++ 310 gbs1632 1 56 ++ 335 gbs1662 322 337 + +++++337 gbs1673 72 90 + +++++ 339 gbs1784 374 395 + 345 gbs1816 136 159 +++++ 348 gbs1899 141 164 + 358 gbs1925 96 157 ++ + 361 gbs2008 1 82 +363 gbs2018 489 556 + +++++ 364 gbs2106 159 183 ++ 371 49 133 + +++++

TABLE 5 Immunogenic proteins identified by bacterial surface display.No. of selected Location of Seq. S. agalactiae clones per identified IDantigenic Putative function ORF and immunogenic (DNA, protein (byhomology) predicted immunogenic aa** screen region (aa) Prot.) gbs0233similar to glycine 4–26, 35–41, 53–61, 73–84, 103–108, 114–120, 140–146,A: 5, B: 9, 1–48, 113–133 463, 475 betaine/carnitine/choline 156–162,192–208, 214–219, 227–233, 239–252, 260–268, H: 3 ABC transporter284–297 (osmoprotectant- binding protein) gbs0419 protein of unknown4–27, 38–44, 50–56, 59–64, 72–79, 83–89, 92–97, 108–116, C: 3, D: 4,1–79, 231–302 464, 476 function/lipoprotein 123–148, 152–167, 183–196,200–220, 232–244, 255–261, J: 2, K: 4 265–274, 282–302, 309–317 gbs0456cell wall surface 6–28, 66–72, 85–105, 115–121, 144–151, 160–170,176–185, A: 9, B: 125, 1–80, 808–821 465, 477 anchor family protein223–230, 252–288, 296–310, 319–333, 367–374, 458–464, F: 9 471–480,483–488, 520–528, 530–549, 559–564, 593–601, 606–616, 636–643, 655–662,676–682, 684–699, 719–726, 735–750, 757–764, 777–785, 799–810, 812–843,846–853, 868–873, 880–889, 891–899, 909–929, 934–940, 963–969, 998–1004,1007–1014, 1016–1022, 1030–1046 gbs0942 protein of unknown 7–24, 35–41,75–81, 91–114, 122–132, 137–144, 148–156, A: 3, B: 9, 1–79, 305–321 466,478 function/lipoprotein 183–192, 194–200, 212–228, 233–238, 251–258,275–295, C: 2, J: 3 326–332, 337–346 gbs0973 gid protein (gid) 31–38,42–52, 66–72, 86–92, 98–104, 115–122, 127–146, D: 2, G: 3, 13–92,135–142 467, 479 154–164, 169–187, 198–212, 225–237, 255–269 H: 8, I: 2,K: 2 gbs0975 Unknown protein 4–36, 39–49, 63–69, 71–77, 81–88, 123–131,133–139, A: 3, B: 5, 1–68, 212–270, 468, 480 160–169, 174–180, 188–194,210–217, 273–278, 289–300, C: 5, D: 2, 402–446 317–334, 336–341,383–401, 425–438 J: 2 gbs1038 permease, putative 21–29, 31–42, 49–63,72–79, 81–93, 112–132, 159–165, B: 2, C: 3, 1–57, 84–106 469, 481188–195, 197–232, 262–267, 279–286, 294–301, 318–326, E: 3, J: 2348–366, 381–405, 409–426, 436–465, 471–480, 484–492, 497–505, 521–544,554–561, 567–577, 581–589, 601–609, 611–622, 636–651, 653–667, 669–685,700–708, 716–722, 729–744, 749–766, 780–786, 789–811, 814–864 gbs1144Unknown protein 6–24, 35–48, 57–63, 72–78, 87–92, 113–119, 123–137, C:30 1–124 470, 482 147–153, 173–181, 212–233 gbs1279 conserved domain13–34, 62–69, 78–83, 86–91, 98–104, 107–115, 146–159, A: 44, B: 2, 1–84471, 483 protein 179–188, 195–205, 209–221, 226–233, 239–253, 276–282,C: 4 284–294, 297–308, 331–354, 375–382, 388–399, 421–433, 449–458,464–469, 472–491, 508–513, 525–531, 534–550, 575–593, 601–618, 629–635,654–661, 666–680, 706–721, 723–740, 771–805, 810–830, 845–851 gbs1441conserved 4–32, 45–64, 73–83, 86–92, 100–111, 125–147, 157–163, A: 2, B:8, 1–71 472, 484 hypothetical protein 170–175, 177–188, 226–232,245–252, 258–274, 320–335, C: 2 348–359 gbs1677 sodium transport 13–40,43–71, 76–83, 87–101, 109–119, 125–156, 162–175, B: 3, C: 2, 1–51 473,485 family protein 182–219, 226–232, 240–262, 270–287, 306–318, 326–342,K: 5 344–408, 414–444, 449–456 gbs2093 Membrane protein, 4–16, 18–34,45–54, 99–108, 134–140, 203–212, 241–257, B: 5, C: 8, 1–87, 342–480 474,486 putative 266–274, 279–291, 308–315, 330–336, 355–370, 374–382, D: 2402–410, 428–455, 466–472, 474–480, 531–554, 560–566, 572–580, 597–618,632–660, 664–674, 676–685, 691–705, 708–735, 750–768

TABLE 6 Immune reactivity of epitopes within identified antigens withhuman sera Peptides Positivity aa from aa to Seq ID gbs0233.1 + 115 132475 gbs0233.2 + 1 26 475 gbs0419.1 ++ 33 55 476 gbs0942.2 +++ 1 25 478gbs0973.1 + 37 61 479 gbs0975.1 + 1 24 480 gbs1038.1 + 1 23 481gbs1144.3 ++ 46 60 482 gbs1279.1 +++ 1 28 483 gbs1279.2 + 23 50 483gbs1279.3 ++ 45 71 483 gbs1441.1 + 1 22 484 gbs1441.2 ++ 17 38 484gbs1677.1 + 1 22 485 gbs1677.2 + 17 38 485 gbs2093.1 + 1 27 486gbs2093.2 ++ 22 47 486 gbs2093.4 + 422 447 486

TABLE 7 Immunogenicity of epitopes in mice Peptide ORF aa from aa toELISA Seq ID gbs0233 115 132 ++++ 475 1 47 ++ gbs0419 1 55 ++++ 476gbs0456 22 85 ++ 477 gbs0942 307 320 + 478 1 44 ++ gbs0973 15 76 ++ 47940 92 +++ gbs0975 1 59 ++ 480 213 269 + 403 445 + gbs1038 1 56 ++++ 48185 105 + gbs1144 37 121 +++++ 482 gbs1279 1 71 +++++ 483 gbs1441 1 38+++ 484 gbs1677 1 38 +++ 485 gbs2093 1 47 +++ 486

REFERENCES

-   Altschul, S., et al. (1990). Journal of Molecular Biology 215:    403-10.-   Balter, S. et al. In Gram positive pathogens ed. by Fischetti V. A.    et al. AMS Press 2000, 154-160.-   Bennett, D., et al. (1995). J Mol Recognit 8: 52-8.-   Brodeur, B., et al. (2000). Infect Immun 68: 5610-8.-   Burnie, J., et al. (1998). J Antimicrob Chemother 41: 319-22.-   Campbell, J., et al. (2000). Obstet Gynecol 96: 498-503.-   Cheng, Q., et al. (2002). Infect Immun 70: 6409-15.-   Clackson, T., et al. (1991). Nature 352: 624-8.-   Devereux, J., et al. (1984). Nucleic acids research 12: 387-95.-   Doherty, E., et al. (2001). Annu Rev Biophys Biomol Struct 30:    457-475.-   Eisenbraun, M., et al. (1993). DNA Cell Biol 12: 791-7.-   Etz, H., et al. (2001). J Bacteriol 183: 6924-35.-   Farley, M. (2001). Clin Infect Dis 33: 556-61.-   Ganz, T. (1999). Science 286: 420-421.-   Georgiou, G. (1997). Nature Biotechnology 15: 29-34.-   Glaser, P., et al. (2002). Mol Microbiol 45: 1499-513.-   Hashemzadeh-Bonehi, L., et al. (1998). Mol Microbiol 30: 676-678.-   Heinje, von G (1987) e.g. Sequence Analysis in Molecular Biology,    Academic Press.-   Hemmer, B., et al. (1999). Nat Med 5: 1375-82.-   Hoe, N., et al. (2001). J Infect Dis 183: 633-9.-   Hornef, M., et al. (2002). Nat Immunol 3:-1033-40.-   Jackson, L., et al. (1995). Ann Intern Med 123: 415-20.-   Johanson, K., et al. (1995). J Biol Chem 270: 9459-71.-   Jones, P., et al. (1986). Nature 321: 522-5.-   Kajava, A., et al. (2000). J Bacteriol 182: 2163-9.-   Kohler, G., et al. (1975). Nature 256: 495-7.-   Larsson, C., et al. (1999). Vaccine 17: 454-8.-   Lewin, A., et al. (2001). Trends Mol Med 7: 221-8.-   Marks, J., et al. (1992). Biotechnology (N Y) 10: 779-83.-   McCafferty, J., et al. (1990). Nature 348: 552-4.-   Michel, J., et al. (1991). Infect Immun 59: 2023-8.-   Navarre, W., et al. (1999). Microbiol Mol Biol Rev 63:174-229.-   Nizet, V. & Rubens, C. E. in Gram positive pathogens ed. by    Fischetti V. A. et al. ASM Press 2000, pp 125-136.-   Okano, H., et al. (1991). J Neurochem 56: 560-7.-   Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression;    CRC Press, Boca Ration, Fla. (1988).-   Paoletti, L., et al. (2002). Semin Neonatol 7: 315-23.-   Paoletti, L., et al. In Gram positive pathogens ed. by    Fischetti V. A. et al. ASM Press 2000, pp 137-153.-   Phillips-Quagliata, J., et al. (2000). J Immunol 165: 2544-55.-   Rammensee, H., et al. (1999). Immunogenetics 50: 213-9.-   Seeger, C., et al. (1984). Proc Natl Acad Sci U S A 81: 5849-52.-   Shibuya, A., et al. (2000). Nature Immunology 1: 441-6.-   Skerra, A. (1994). Gene 151: 131-5.-   Tang, D., et al. (1992). Nature 356:152-4.-   Tempest, P., et al. (1991). Biotechnology (N Y) 9: 266-71.-   Tettelin, H., et al. (2002). Proc Natl Acad Sci U S A 99: 12391-6.-   Tourdot, S., et al. (2000). Eur 1 Immunol 30: 3411-21.-   Wiley, J., et al. (1987) Current Protocols in Molecular Biology.

1. A composition comprising an isolated hyperimmune serum-reactiveantigen fragment consisting of an amino acid sequence of 399-417,503-519, 544-563, 489-556, 386-458, or 458-624 of SEQ ID NO:364.
 2. Thecomposition of claim 1, wherein the hyperimmune serum-reactive antigenfragment binds to anti-S. agalactiae serum.
 3. The composition of claim1, further defined as comprising at least two different hyperimmuneserum-reactive antigen fragments.
 4. The composition of claim 3, whereinthe at least two different hyperimmune serum-reactive antigen fragmentsare both bind to anti-S.agalactiae serum.
 5. The composition of claim 1,further comprising an immunostimulatory substance.
 6. The composition ofclaim 5, wherein the immunostimulatory substance is a polycationicpolymer, an immunostimulatory non-methylated deoxynucleotide (ODN), apeptide containing at least two LysLeuLys motifs alum, or a Freund'scomplete or incomplete adjuvant.
 7. A pharmaceutical compositioncomprising: (a) an isolated hyperimmune serum-reactive antigen fragmentconsisting of an amino acid sequence of 399-417, 503-519, 544-563,489-556, 386-458, or 458-624 of SEQ ID NO:364; and (b) animmunostimulatory substance.